Fragments of prion proteins

ABSTRACT

Synthetic polypeptides having at least one antigenic site of a prior protein, methods for their use and manufacture, antibodies raised against such polypeptides and diagnostic kits containing these polypeptides or antibodies.

RELATED APPLICATIONS

[0001] This application is a divisional of U.S. Ser. No. 09/076,721filed May 13, 1998, which is a divisional of Ser. No. 08/244,701 filedJun. 2, 1994, now U.S. Pat. No. 5,773,572 issued Jun. 30, 1998, which isa §371 of PCT/GB92/02246 filed Dec. 3, 1992.

[0002] The present invention relates to synthetic polypeptides. Inparticular it relates to synthetic polypeptides which emulate thethree-dimensional structures and/or electrostatic surfaces and/or otherphysical, chemical and structural properties of specific regions ofproteins thought to be involved in the molecular pathology of spongiformencephalopathies. It is of particular interest to the design ofimmunodiagnostics, vaccines and other medical, veterinary or scientificagents in relation to human, bovine and ovine spongiformencephalopathies.

[0003] Spongiform encephalopathies are a group of degenerativeneurological diseases. Examples have been found in a number of speciesincluding sheep (where it is known as scrapie), cows (BSE) and humans(Creutzfeldt-Jakob disease (CJD) and kuru) (Review article, Taylor, D.M. Veterinary Record 125, 413-415 (1989)). Similar conditions have alsobeen found in the wild mink population and in captive kudus (a kind ofantelope) and tigers. It has been variously reported that BSE can betransmitted under laboratory conditions to mice and pigs. This crossingof species barriers by the infective agent has led to increased concernthat transfer to humans could occur.

[0004] These diseases are characterized by a slow incubation time offour to five years after which the clinical symptoms of progressivedegeneration of mental state, including aggressiveness and lack ofcoordination, appear. Post mortems reveal a characteristic pattern ofvacuolation in brain tissue due to the destruction of neural cells, andthe deposition of unusual protein fibers.

[0005] Although the form of the disease found in sheep (scrapie) hasbeen known for many years, spongiform encephalopathies have come toprominence within the last decade following the appearance of BSE incattle farms. The incidence of BSE in the United Kingdom has increasedmarkedly during this period and public concern over the possibletransmission of the disease to humans has led to a collapse in the beefmarket. Thus for both veterinary and economic reasons, there is anurgent need for diagnostic agents to detect infection and for vaccinesto prevent infection.

[0006] It is believed that the causative agent of scrapie and itscounterparts in other animals is a so-called “prion”, that is aninfective particle comprising protein only and no nucleic acid, thepresence of the latter being required in the case of a conventionalvirus. In scrapie, one particular protein (termed prion protein,PrP^(SC)) has been found to co-purify with infectivity and can produce ascrapie-like condition in brain cell cultures from other animals, suchas hamsters, under laboratory conditions. PrP^(SC) is the only knowncomponent of the characteristic protein fibers deposited in the braintissue of scrapie-infected sheep. The term “PrP^(SC)” as used hereinshould be taken to refer not only to the specific Prion proteinidentified in sheep but also to those homologous proteins found in manyother species which appear to undergo a structural modification asdescribed hereinafter. The term PrP^(C) “shall be used in respect of thenormal cellular counterpart to PrP^(SC).

[0007] The major problem in the search for a specific diagnostic agentor synthetic vaccine against the scrapie agent PrP^(SC) is that it isalmost identical to the natural form of the protein PrP^(C). The naturalfunction of this protein is not yet understood but the remarkably strongconservation of primary structure between homologous proteins fromdifferent species suggests that it has an essential structural orfunctional role within the organism.

[0008] In spite of the almost identical form of these prions to thenatural proteins, we have deduced synthetic peptide structurescomprising at least one antigenic property, such as an epitopic site andthese synthetic peptides may be used to produce diagnostic agents andvaccines.

[0009] The responses of the B and T cells of the immune system are notspecified by a global recognition of a whole protein but rather byrecognition of a small region of the protein surface known as epitopicsite. Such sites may be formed by a continuous section of peptide chainor may be discontinuous, where separated sections of peptide chain arebrought together at the protein surface due to folding of the chain. Oneaim in producing a synthetic peptide vaccine is to mimic the structureof a particular epitope and thereby cause a primary immune responseleading to the production of memory B cells which will secreteantibodies on subsequent exposure to the parent protein so producing agreatly enhanced response to secondary infection. A similar mechanismvia priming of the cytotoxic T cells to respond more vigorously to aparticular antigen will also occur.

[0010] However, problems exist with the application of traditionalmethods of vaccine production to this disease as it is believed that themolecular structure of the protein prion rather than nucleic acidsequence passes on infectivity in the prion. The usual method of viralvaccine production involves the inactivation of the virus in some way todestroy infectivity while preserving epitopic sites. Such techniques asheat treatment or serial passaging of the virus through a culture areused, but these approaches would not lead to a loss of infectivity of aprion unless conditions were such as to cause protein denaturation. Ifthe conditions are severe enough to inactivate the prion protein thendenaturation of the protein occurs and any epitopic sites are lost. Thusthere is a major problem in trying to obtain antigenic but non-infectiveprion proteins by conventional routes. It is known, for example, thatthe scrapie agent in sheep is particularly resistant to chemical orphysical inactivation (Hodgson, J. Bio/Technology 8 990 (1990)).

[0011] In one aspect our invention provides a synthetic polypeptidehaving at least one antigenic site of a prion protein. Preferably theprion protein is of a form which only exists in nervous tissue of amammal suffering from spongiform encephalopathy.

[0012] We have found that prion proteins of the type mentioned abovecomprise six regions of interest, labeled A to F, and two related frameshift peptide sequences, viz:1) a repeating section in region E havingundergone a nucleic acid coding sequence frame shift of +1 (FSa) and 2)the repeating section in region E having undergone a nucleic acid codingsequence frame shift of −1 (FSb).

[0013] With regard to region A, our invention provides a syntheticpeptide sequence according to general formula (I): Seq. I.D. No: 52X-(R₁-Lys-His-R₂)-Ala-Gly-Ala-Ala-Ala-R₃-Gly-Ala-Val-Val-Gly-Gly-Leu-Gly-Gly-Tyr-Met-Leu-Gly-Ser-Ala-Met-Ser-(Arg-Pro-R₄-R₅)-Y (I)

[0014] wherein R₁ is an amino acid residue selected from Met, Leu andPhe;

[0015] R₂ is either Met or Val;

[0016] R₃ is Ala or is absent;

[0017] R₄ and R₅ are independently an amino acid residue selected fromLeu, Ile and Met; one or more residues within brackets may be present orabsent with the proviso that if they are present they are attached tothe rest of the peptide in sequence; and X and Y may each independentlybe absent or independently be one or more additional amino acidresidues.

[0018] It will be apparent for example that the residues at theN-terminal of the sequence may be present as “R₂”- or “His-R₂-,” or“Lys-His-R₂-” or “R₁-Lys-His-R₂-.” Similarly, the preferable residues atthe C-terminal may be present as “-Arg”, or “-Arg-Pro,” or“-Arg-Pro-R₄,” or “-Arg-Pro-R₂-R₅.”

[0019] Preferably, R₁, if present, is Met, R₃, is Ala and R₅, ifpresent, is Ile. Also, if R₄ is Met then R₄, if present, is Ile. Beloware preferred sequences (Seq. I.D. No: 1 and Seq. I.D. No: 2) of formulaI relating to bovine and ovine and to human prion proteins respectively:Seq. I.D. No: 1 X-(Met-Lys-His-Val)-Ala-Gly-Ala-Ala-Ala-Ala-Gly-Ala-Val-Val-Gly-Gly-Leu-Gly-Gly-Tyr-Met-Leu-Gly-Ser-Ala-Met-Ser-(Arg-Pro-Leu-Ile)-Y; and Seq. I.D. No: 2X-(Met-Lys-His-Met)-Ala-Gly-Ala-Ala-Ala-Ala-Gly-Ala-Val-Val-Gly-Gly-Leu-Gly-Gly-Tyr-Met-Leu-Gly-Ser-Ala-Met-Ser-(Arg-Pro-Ile-Ile)-Y

[0020] A particularly preferred sequence according to formula I is Seq.I.D. No: 51: Lys-His-Met-Ala-Gly-Ala-Ala-Ala-Ala-Gly-Ala-Val-Val-Gly-Gly-Leu-Gly-Gly-Tyr-Met-Leu-Gly-Ser-Ala- Met-Ser-Arg-Gly-Cys.

[0021] Naturally, our invention encompasses significant sub-fragments ofthe sequence according to formula I above and preferred sub-fragmentsare: Seq. I.D. No: 63 i) X-(His-R₂-Ala-Gly)-Ala-Ala-Ala-R₃-Gly-Ala-Val-Val-(Gly-Gly-Leu-Gly)-Y and; Seq. I.D. No: 64 ii)X-(Gly-Gly-Leu-Gly)-Gly-Tyr-Met-Leu-Gly-Ser-Ala-Met-Ser-(Arg-Pro-R₄-R₅)-Y

[0022] wherein R₂, R₃, R₄, R₅, X and Y are as defined for formula I andone or more residues in brackets may be absent or present as in formulaI.

[0023] It will be clear from the foregoing that preferred sub-fragmentsrelating to both bovines and ovines are Seq. I.D. No: 3 i)X-(His-Val-Ala-Gly)-Ala-Ala-Ala-Ala-Gly-Ala-Val-Val-Gly-(Gly-Leu-Gly-Gly-Y; and Seq. I.D. No. 4 ii)(Gly-Gly-Leu-Gly)-Gly-Tyr-Met-Leu-Gly-Ser-Ala-Met-Ser-(Arg-Pro-Leu-Ile)-Y.

[0024] Similarly, preferred sub-fragments for humans are: Seq. I.D. No:5 i) X-(His-Met-Ala-Gly)-Ala-Ala-Ala-Ala-Gly-Ala-Val-Val-Gly-(Gly-Leu-Gly-Gly)-Y; and Seq. I.D. No: 6 ii)X-(Gly-Gly-Leu-Gly)-Gly-Tyr-Met-Leu-Gly-Ser-Ala-Met-Ser-(Arg-Pro-Ile-Ile)-Y.

[0025] With regard to region B, our invention provides a syntheticpeptide sequence according to general formula II: Seq. I.D. No: 53X-(Ser-Ala-Met-Ser)-Arg-Pro-R₄-R₅-His-Phe-Gly-R₆-Asp-R₇-Glu-Asp-Arg-Tyr-Tyr-Arg-Glu-Asn-Met-R₈-Arg- (Tyr-Pro-Asn-Gln)-Y(II)

[0026] wherein R₄ and R₅ are the same as in formula I;

[0027] R₆ is either Asn or Ser;

[0028] R₇ is either Tyr or Trp;

[0029] R₈ is an amino acid residue selected from His, Tyr and Asn;

[0030] one or more residues within brackets may be present or absentwith the proviso that if they are present they are attached to the restof the peptide in sequence; and X and Y may each independently be absentor independently be one or more additional amino acid residues.

[0031] Preferably in a sequence according to formula II, R₅ is Ile, R₇is Tyr and R₈ is His or Tyr. Below are preferred sequences of formula IIrelating to bovine, ovine and human prion proteins respectively: Seq.I.D. No: 7 X-(Ser-Ala-Met-Ser)-Arg-Pro-Leu-Ile-His-Phe-Gly- +00Ser-Asp-Tyr-Glu-Asp-Arg-Tyr-Tyr-Arg-Glu-Asn-Met- +00His-Arg-(Tyr-Pro-Asn-Gln)-Y; +00 Seq. I.D. No: 8X-(Ser-Ala-Met-Ser)-Arg-Pro-Leu-Ile-His-Phe-Gly- +00Asn-Asp-Tyr-Glu-Asp-Arg-Tyr-Tyr-Arg-Glu-Asn-Met- +00Tyr-Arg-(Tyr-Pro-Asn-Gln-Y; and +00 Seq. I.D. No: 9X-(Ser-Ala-Met-Ser)-Arg-Pro-Ile-Ile-His-Phe-Gly- +00Ser-Asp-Tyr-Glu-Asp-Arg-Tyr-Tyr-Arg-Glu-Asn-Met- +00His-Arg-(Tyr-Pro-Asn-Gln)-Y.

[0032] Particularly preferred sequences are selected from Seq. I. D. No:42 Seq. I.D. No: 42 Ser-Ala-Met-Ser-Arg-Pro-Leu-Ile-His-Phe-Gly-Asn-Asp-Tyr-Glu-Asp-Arg-Tyr-Tyr-Gly-Cys; and Seq. I.D. No: 43Ser-Ala-Met-Ser-Arg-Pro-Leu-Ile-His-Phe-Gly-Ser-Asp-Tyr-Glu-Asp-Arg-Tyr-Tyr-Gly-Cys.

[0033] Again it will be apparent that our invention encompassessignificant sub-fragments of the sequence according to Formula II and apreferred general sub-fragment has the sequence: Seq. I.D. No: 65X-(Ser-Ala-Met-Ser)-Arg-Pro-R₄-R₅-His-Phe-Gly-R₆-Asp-R₇-Glu-Asp-Arg-Tyr-Tyr-(Arg-Glu-Asn-Met)-Y

[0034] R₄ to R₇, X and Y are as defined in formula II and one or moreresidues in brackets may be present or absent. Preferably, R₅ is Ile andR₇ is Tyr. It will be appreciated that preferred sub-fragments relatingto bovines, ovines and humans are respectively;X-(Ser-Ala-Met-Ser)-Arg-Pro-Leu-Ile-His-Phe-Gly-Ser-Asp-Tyr-Glu-Asp-Seq. I.D. No:10 Arg-Tyr-Tyr-(Arg-Glu-Asn-Met)-Y;X-(Ser-Ala-Met-Ser)-Arg-Pro-Leu-Ile-His-Phe-Gly-Asn-Asp-Tyr-Glu-Asp-Seq. I.D. No:11 Arg-Tyr-Tyr-(Arg-Glu-Asn-Met)-Y; andX-(Ser-Ala-Met-Ser)-Arg-Pro-Ile-Ile-His-Phe-Gly-Ser-Asp-Tyr-Glu-Asp-Seq. I.D. No:12 Arg-Tyr-Tyr-(Arg-Glu-Asn-Met)-Y.

[0035] Our invention provides in respect of region C a synthetic peptidesequence according to general formula III:X-(Asn-Met-R₈-Arg)-Tyr-Pro-Asn-Gln-Val-Tyr-Tyr-Arg-Pro-R₉-Asp-R₁₀- Seq.I.D. No:54 Tyr-R₁₁-Asn-Gln-Asn-Asn-Phe-Val-His-(Asp-Cys-Val-Asn)-Y

[0036] wherein R₈ is an amino acid residue selected from His, Tyr andAsn;

[0037] R₉ is Val or Met;

[0038] R₁₀ is an amino acid residue selected from Gln, Glu and Arg;

[0039] R₁₁ is Ser or Asn; one or more residues within brackets may bepresent or absent with the proviso that if they are present they areattached to the rest of the peptide in sequence and X and Y may eachindependently be absent or independently be one or more additional aminoacid residues.

[0040] Preferably in a sequence according to formula III, R₈ is His orTyr and R₁₁ is Ser. Below are preferred sequences of formula IIIrelating to bovine, ovine and human prion proteins respectively:X-(Asn-Met-His-Arg)-Tyr-Pro-Asn-Gln-Val-Tyr-Tyr-Arg-Pro-Val-Asp-Gln-Seq. I.D. No:13 Tyr-Ser-Asn-Gln-Asn-Asn-Phe-Val-His-(Asp-Cys-Val-Asn)-Y;X-(Asn-Met-Tyr-Arg)-Tyr-Pro-Asn-Gln-Val-Tyr-Tyr-Arg-Pro-Val-Asp-Arg-Seq. I.D. No:14 Tyr-Ser-Asn-Gln-Asn-Asn-Phe-Val-His-(Asp-Cys-Val-Asn)-Y;and X-(Asn-Met-His-Arg)-Tyr-Pro-Asn-Gln-Val-Tyr-Tyr-Arg-Pro-Met-Asp-Seq. I.D. No:15Glu-Tyr-Ser-Asn-Gln-Asn-Asn-Phe-Val-His-(Asp-Cys-Val-Asn)-Y.

[0041] Particularly preferred sequences are selected from Seq. I.D. No:44 Asn-Met-Tyr-Arg-Tyr-Pro-Asn-Gln-Val-Tyr-Tyr-Arg-Pro-Val-Asp-Arg-Tyr-Seq. I.D. No:44 Ser-Asn-Gln-Asn-Asn-Phe-Val-His-Gly-Cys; andAsn-Met-His-Arg-Tyr-Pro-Asn-Gln-Val-Tyr-Tyr-Arg-Pro-Val-Asp-Gln-Tyr-Seq. I.D. No:45 Ser-Asn-Gln-Asn-Asn-Phe-Val-His-Gly-Cys.

[0042] Significant sub-fragments of the sequence according to formulaIII form part of this invention and a preferred sub-fragment has thesequence:X-(Arg-Tyr-Pro-Asn)-Gln-Val-Tyr-Tyr-Arg-Pro-R₉-Asp-R₁₀-Tyr-R₁₁-Asn- Seq.I.D. No:66 Gln-Asn-Asn-Phe-Val-His-(Asp-Cys-Val-Asn)-Y.

[0043] Preferred sub-fragments relating to bovines, ovines and humansare respectively:X-(Arg-Tyr-Pro-Asn)-Gln-Val-Tyr-Tyr-Arg-Pro-Val-Asp-Gln-Tyr-Ser-Asn-Seq. ID. No:16 Gln-Asn-Asn-Phe-Val-His-(Asp-Cys-Val-Asn)-Y;X-(Arg-Tyr-Pro-Asn)-Gln-Val-Tyr-Tyr-Arg-Pro-Val-Asp-Arg-Tyr-Ser-Asn-Seq. I.D. No:17 Gln-Asn-Asn-Phe-Val-His-(Asp-Cys-Val-Asn)-Y; andX-(Arg-Tyr-Pro-Asn)-Gln-Val-Tyr-Tyr-Arg-Pro-Met-Asp-Glu-Tyr-Ser-Asn-Seq. I.D. No:18 Gln-Asn-Asn-Phe-Val-His-(Asp-Cys-Val-Asn)-Y.

[0044] In respect of region D, our invention provides a syntheticpeptide sequence according to general formula IV:X-(Tyr-Tyr-R₁₂-R₁₃-Arg)-R₁₄-R₁₅-Ser-R₁₆-R₁₇-R₁₈-Leu-Phe-Ser-Ser-Pro-Pro-Seq. I.D. No:55Val-Ile-Leu-Leu-IIe-Ser-Phe-Leu-Ile-Phe-Leu-R₁₉-Val-Gly-Y (IV)

[0045] wherein R₁₂ is Asp or Gln;

[0046] R₁₃ is Gly or absent;

[0047] R₁₄ is Gly or Arg;

[0048] R₁₅ is Ala or Ser;

[0049] R₁₆ is Ser or absent;

[0050] R₁₇ is an amino acid residue selected from Ala, Thr, Met and Val;

[0051] R₁₈ is Val or Ile;

[0052] R₁₉ is Ile or Met; one or more residues within brackets may bepresent or absent with the proviso that if they are present they areattached to the rest of the peptide in sequence and X and Y may eachindependently be absent or independently be one or more additional aminoacid residues.

[0053] Preferably in a sequence according to formula IV R₁₂ is Gln, R₁₃is absent, R₁₄ is Gly, R₁₆ is absent, R₁₇ is Val or Met and R₁₉ is Ile.

[0054] Preferred sequences of formula IV relating to bovine and ovineand to human prion proteins respectively are given below:X-(Tyr-Tyr-Gln-Arg)-Gly-Ala-Ser-Val-Ile-Leu-Phe-Ser-Ser-Pro-Pro-Val-Ile-Seq. I.D. No:19 Leu-Leu-Ile-Ser-Phe-Leu-Ile-Phe-Leu-Ile-Val-Gly-Y; andX-(Tyr-Tyr-Gln-Arg)-Gly-Ser-Ser-Met-Val-Leu-Phe-Ser-Ser-Pro-Pro-Val-Seq. I.D. No:20 Ile-Leu-Leu-Ile-Ser-Phe-Leu-Ile-Phe-Leu-Ile-Val-Gly-Y.

[0055] Clearly, it will be recognized that the present inventionincludes with its ambit significant sub-fragments of the sequenceaccording to formula IV and a preferred general sub-fragment has thesequence:X-(R₁₄-R₁₅-Ser-R₁₆-R₁₇)-R₁₈-Leu-Phe-Ser-Ser-Pro-Pro-Val-Ile-(Leu-Leu-Ile-Seq. I.D. No:67 Ser)-Y

[0056] Wherein R₁₄ to R₁₈, X and Y are as defined in formula IV and oneor more residues within brackets may be present or absent as in formulaIV.

[0057] It is preferred that in a sub-fragment as given above, R₁₄ isGly, R₁₆ is absent and R₁₇ is Val or Met. Below are preferredsub-fragments relating to bovines and ovines and to humans respectively:X-(Gly-Ala-Ser-Val)-Ile-Leu-Phe-Ser-Ser-Pro-Pro-Val-Ile-(Leu-Leu-Ile-Seq. I.D. No:21 Ser)-Y; andX-(Gly-Ser-Ser-Met)-Val-Leu-Phe-Ser-Ser-Pro-Pro-Val-Ile-(Leu-Leu-Ile-Seq. I.D. No:22 Ser)-Y.

[0058] Our invention provides in respect of Region E three syntheticpolypeptide sequences according to general formulae Va, Vb and Vc: Seq.I.D. No: 56 X-(Pro-Gly-Gly-R₂₀)-Trp-Asn-Thr-Gly-Gly-Ser-Arg-Tyr-Pro-Gly-Gln-Gly-Ser-Pro-Gly-Gly-Asn-Arg-Tyr-Pro-Pro-Gln-Gly-(Gly-R₂₁-R₂₂-Trp)-Y (Va); Seq. I.D. No: 57X-(Gly-Gly-R₂₁-R₂₂-Trp)-Gly-Gln-Pro-His-Gly-Gly- Gly-R₂₃-Trp(Gly-Gln-Pro-His)-Y(Vb); and Seq. I.D. No: 58X-(Gly-Gly-Gly-Trp)-Gly-Gln-Gly-Gly-R₂₄-R₂₅-His-R₂₆-Gln-Trp-Asn-Lys-Pro-R₂₇-Lys-Pro-Lys-Thr-R₂₈- R₂₉-Lys(-His-R₃₀-Ala-Gly)-Y (Vc)

[0059] Wherein R₂₀, R₂₁, R₂₃ and R₂₄ are each independently either Glyor absent,

[0060] R₂₂ either Gly or Thr;

[0061] R₂₅ is either Thr or Ser;

[0062] R₂₆ is an amino acid residue selected from Gly, Ser and Asn;

[0063] R₂₇ and R₂₈ are each independently either Asn or Ser;

[0064] R₂₉ is an amino acid residue selected from Met, Leu and Phe;

[0065] R₃₀ is either Val or Met; one or more residues within bracketsmay be present or absent with the proviso that if they are present theyare attached to the rest of the peptide in sequence; and X and Y mayeach independently be absent or independently be one or more additionalamino acid residues.

[0066] With regard to formulae Va to Vc above, it is preferred that R₂₂is Gly, R₂₃ is absent, R₂₆ is Gly or R₂₇ is Ser, R₂₈ is Asn and R₂₉ isMet.

[0067] Preferred bovine sequences of prion proteins according toformulae Va to Vc are given below: Seq. I.D. No: 23X-(Pro-Gly-Gly-Gly)-Trp-Asn-Thr-Gly-Gly-Ser-Arg-Tyr-Pro-Gly-Gln-Gly-Ser-Pro-Gly-Gly-Asn-Arg-Tyr-Pro-Pro-Gln-Gly-(Gly-Gly-Gly-Trp)-Y; Seq. I.D. No: 24X-(Gly-Gly-Gly-Trp)-Gly-Gln-Pro-His-Gly-Gly-Gly-Trp-(Gly-Gln-Pro-His)-Y; and Seq. I.D. No: 25X-(Gly-Gly-Gly-Trp)-Gly-Gln-Gly-Gly-Thr-His-Gly-Gln-Trp-Asn-Lys-Pro-Ser-Lys-Pro-Lys-Thr-Asn-Met-Lys(-His-Val-Ala-Gly)-Y.

[0068] Preferred sequences of formulae Va to Vc relating to ovine prionproteins are as follows: Seq. I.D. No: 26X-(Pro-Gly-Gly-Gly)-Trp-Asn-Thr-Gly-Gly-Ser-Arg-Tyr-Pro-Gly-Gln-Gly-Ser-Pro-Gly-Gly-Asn-Arg-Tyr-Pro-Pro-Gln-Gly-(Gly-Gly-Gly-Trp)-Y; Seq. I.D. No: 27X-(Gly-Gly-Gly-Trp)-Gly-Gln-Pro-His-Gly-Gly-Gly- Trp-(Gly-Gln-Pro-His)Y;and Seq. I.D. No: 28 X-(Gly-Gly-Gly-Trp)-Gly-Gln-Gly-Gly-Ser-His-Ser-Gln-Trp-Asn-Lys-Pro-Ser-Lys-Pro-Lys-Thr-Asn-Met-Lys(-His-Val-Ala-Gly)-Y.

[0069] Preferred sequences of formulae Va to Vc relating to human prionproteins are as follows: Seq. I.D. No: 29X-Pro-Gly-Gly-Gly-Trp-Asn-Thr-Gly-Gly-Ser-Arg-Tyr-Pro-Gly-Gln-Gly-Ser-Pro-Gly-Gly-Asn-Arg-Tyr-Pro-Pro-Gln-Gly-(Gly-Gly-Gly-Trp)-Y; Seq. I.D. No: 30X-(Gly-Gly-Gly-Trp)-Gly-Gln-Pro-His-Gly-GIy-Gly-Trp-(Gly-Gln-Pro-His)-Y; and Seq. I.D. No: 31X-(Gly-Gly-Gly-Trp)-Gly-Gln-Gly-Gly-Gly-Thr-His-Ser-Gln-Trp-Asn-Lys-Pro-Ser-Lys-Pro-Lys-Thr-Asn- Met-Lys(-His-Met-Ala-Gly)-Y.

[0070] Particularly preferred sequences of formulae Va to Vc consist of:Seq. I.D. No: 49 Gly-Gly-Trp-Asn-Thr-Gly-Gly-Ser-Arg-Tyr-Pro-Gly-Gln-Gly-Ser-Pro-Gly-Gly-Asn-Arg-Tyr-Pro-Pro-Gln- Gly-Gly-Gly-Cys; Seq.I.D. No: 46 Gly-Gln-Pro-His-Gly-Gly-Gly-Trp-Gly-Gln-Pro-His-Gly-Gly-Gly-Trp-Gly-Gln-Pro-His-Gly-Gly-Gly-Trp- Gly-Cys; and Seq. I.D.No: 47 Gly-Gln-Gly-Gly-Ser-His-Ser-Gln-Trp-Asn-Lys-Pro-Ser-Lys-Pro-Lys-Thr-Asn-Met-Lys-His-Val-Gly-Cys.

[0071] We have noted that in the nucleic acid sequence corresponding toregion E, it is possible for the repeating sequence of formula Vb tohave undergone a frame shift of either +1 or −1. Such frame shifts giverise to altered sequences in region E of the prion protein and ourinvention provides a synthetic polypeptide having a sequence wherein arepeat in region E has undergone a −1 frame shift as given in formulaVI: Seq. I.D. No: 59 X-(R₃₁-R₃₂-Trp-R₃₃)-Trp-Leu-Gly-R₃₄-R₃₅-R₃₆-Trp-R₃₇ (Trp-Leu-Gly-R₃₈)-Y(VI)

[0072] Wherein R₃₁ and R₃₅ are each independently either Ala or Thr; R₃₂and R₃₆ are each independently an amino acid residue selected from Ser,Pro and Thr;

[0073] R₃₃ and R₃₇ are each independently either Trp or Arg;

[0074] R₃₄ and R₃₈ are each independently an amino acid residue selectedfrom Ala, Ser, Pro and Thr; one or more residues within brackets may bepresent or absent with the proviso that if they are present they areattached to the rest of the peptide in sequence; and X and Y may eachindependently be absent or independently be one or more, additionalamino acid residues.

[0075] With regard to −1 frame shifts in respect of region E in bovines,it is preferred that R₃₁ is Ala, R₃₂, R₃₄, R₃₆ and R₃₈ are eachindependently either Ser or Pro, R₃₃ and R₃₇ are Arg and R₃₅ 35 is Ala.

[0076] It should be noted that preferred sequences for −1 frame shiftsin region E of ovines differ in some respects to those given for bovinesand in a referred ovine sequence R₃₁, R₃₂, R₃₃, R₃₅, R₃₆ and R₃₇correspond to the definitions given for formula VI above; and R₃₄ andR₃₈ are each independently selected from Ser, Pro and Thr.

[0077] In a preferred human sequence according to formula VI R₃₁, R₃₄,R₃₅ and R₃₈ are each Ala, R₃₂ and R₃₆ are each independently either Seror Pro and R₃₃ and R₃₇ are both Trp.

[0078] As mentioned previously, the frame shift may be +1 in the repeatportion of region E and this gives rise to different amino acidsequences. Accordingly, our invention provides a synthetic polypeptideaccording to formula VII below which relates to a +1 frame shift in therepeat of region E: Seq. I.D. No: 60X-(R₃₉-R₄₀-Met-R₄₁)-Val-Ala-Gly-R₄₂-R₄₃-R₄₄-Met-R₄₅-(Val-Ala-Gly-R₄₆)-Y(VII)

[0079] Wherein R₃₉ and R₄₃ are each independently either Ser or Asn; R₄₀and R₄₄ are each independently an amino acid residue selected from Pro,Leu and His, R₄, and R₄₅ are each independently Val or Glu; R₄₂ and R₄₆are each independently selected from Val, Ala, Asp and Gly; one or moreresidues within brackets may be present or absent with the proviso thatif they are present they are attached to the rest of the peptide insequence; and X and Y may each independently be absent or independentlybe one or more, additional amino acid residues.

[0080] A preferred bovine sequence according to formula VII comprisesR₃₉ and R₄₃ each being Ser, R₄₂ and R₄₆ each being independently eitherVal or Ala and R₄₄ being either Pro or Leu; with the other R groupsbeing as defined in formula VII.

[0081] A preferred sequence according to formula VII relating to ovinesis the same as given in general formula VII except R₄₂ and R₄₆ are eachindependently selected from Val, Ala and Asp.

[0082] With regard to a preferred human sequence according to formulaVII, R₃₉ and R₄₃ are Ser, R₄₀ and R₄₄ are each independently Pro or Leu,R₄₁ and R₄₅ are Val and R₄₂ and R₄₆ are each independently either Asp orGly.

[0083] Our invention also provides a synthetic peptide sequence relatingto region F and having either the general formula VIIIa or VIIIb: Seq.I.D. No: 61 X-(Asn-Phe-Val-His)-Asp-Cys-Val-Asn-Ile-Thr-R₄₇-Lys-R₄₈-His-Thr-Val-R₄₉-Thr-Thr-Thr-Lys-Gly-Glu-Asn-Phe-Thr-Glu-(Thr-Asp-R₅₀-Lys)-Y(VIIIa) Seq. I.D. No: 62X-(Met-Cys-R₅₁-Thr)-Gln-Tyr-R₅₂-R₅₃-Glu-Ser-Gln-Ala-Tyr-Tyr-R₅₄-R₅₅-Arg-(R₅₆-R₅₇-Ser-R₅₈- R₅₉)-Y(VIIIb)

[0084] Wherein R₄₇ is either Ile or Val;

[0085] R₄₈ and R₅₂ are each independently either Gln or Glu;

[0086] R₄₉ is either Val or Thr;

[0087] R₅₀ is either Val or Ile;

[0088] R₅₁ is an amino acid residue selected from Ile, Thr and Val;

[0089] R₅₂ is Gln or Glu;

[0090] R₅₃ is either Arg or Lys;

[0091] R₅₄ is either Asp or Gln;

[0092] R₅₅ is Gly or is absent;

[0093] R₅₆ is either Gly or Arg;

[0094] R₅₇ is either Ala or Ser;

[0095] R₅₈ is Ser or absent;

[0096] R₅₉ is an amino acid residue selected from Ala, Thr, Met and Val;

[0097] one or more residues within brackets may be present or absentwith the proviso that if they are present they are attached to the restof the peptide in sequence; and X and Y may each independently be absentor independently be one or more, e.g. 3, additional amino acid residues.

[0098] It is preferred in formula VIIIa that R₄₉ is Thr and in formulaVIIIb that R₅₁ is Ile, R₅₃ is Arg, R₅₄ is Gln, R₅₅ is absent, R₅₆ isGly, R₅₇ is Ala and R₅₈ is absent.

[0099] Most preferred bovine, ovine and human sequences according toformulae VIIIa and VIIIb are given below in order: Seq. I.D. No: 32X-(Asn-Phe-Val-His)-Asp-Cys-Val-Asn-Ile-Thr-Val-Lys-Glu-His-Thr-Val-Thr-Thr-Thr-Thr-Lys-Gly-Glu-Asn-Phe-Thr-Glu-(Thr-Asp-Ile-Lys)-Y bovine (VIIIa); and Seq. I.D. No: 33X-(Met-Cys-Ile-Thr)-Gln-Tyr-Gln-Arg-Glu-Ser-Gln-Ala-Tyr-Tyr-Gln-Arg-(Gly-Ala-Ser-Val)-Y bovine (VIIIb); Seq. I.D. No: 34X-(Asn-Phe-Val-His)-Asp-Cys-Val-Asn-Ile-Thr-Val-LyS-Gln-His-Thr-Val-Thr-Thr-Thr-Thr-Lys-Gly-Glu-Asn-Phe-Thr-Glu-(Tbr-ASp-Ile-LyS)-Y ovine (VIIIa), and Seq. I.D. No: 35X-(Met-Cys-Ile-Thr)-Gln-Tyr-Gln-Arg-Glu-Ser-Gln-Ala-Tyr-Tyr-Gln-Arg-(Gly-Ala-Ser-Val)-Y ovine (VIIIb) Seq. I.D. No: 36X-(Asn-Phe-Val-His)-Asp-Cys-Val-Asn-Ile-Thr-Ile-Lys-Gln-HiS-Thr-Val-Thr-Thr-Thr-Thr-Lys-Gly-Glu-Asn-Phe-Thr-G1u-(Thr-Asp-Val-Lys)-Y human (VIIIa), and Seq. I.D. No: 37X-(Met-Cys-Ile-Thr)-Gln-Tyr-Glu-Arg-Glu-Ser-Gln-Ala-Tyr-Tyr-Gln-Arg-(Gly-Ser-Ser-Met)-Y human (VIIIb).

[0100] Particularly preferred sequences according to formula VIIIa andVIIIb are selected from Seq. I.D. No: 50Val-Asn-Ile-Thr-Val-Lys-Gln-His-Thr-Val-Thr-Thr-Thr-Thr-Lys-Gly-Glu-Asn-Phe-Thr-Glu-Gly-Cys; and Seq. I.D. No: 48Cys-Ile-Thr-Glu-Tyr-Gln-Arg-Glu-Ser-Gln-Ala-Tyr- Tyr-Gln-Arg.

[0101] Synthetic polypeptides according to any one of formulae I toVIIIb above without X and Y being present will of course be useful, forexample, in the production of antibodies. However, when X or Y arepresent they may be any length but preferably less than 20 amino acids,more preferably less than 10, e.g. 3 to 6. It will of course beappreciated that a sequence according to any one of formulae I to VIIIbmay constitute a protein with X and Y being major portions of theprotein with the antigenic sequence being for example, part of anexposed loop on a globular protein.

[0102] It is preferred that if X or Y are present they are relativelyshort sequences, typically 1 to 3 residues long. In most instances X ispreferably absent and Y is 1 or 2 residues long, e.g. -Cys or -Gly-Cys.

[0103] All the sequences herein are stated using the standard I.U.P.A.C.three-letter-code abbreviations for amino acid residues defined asfollows: Gly-Glycine, Ala-Alanine, Val-Valine, Leu-Leucine,Ile-Isoleucine, Ser-Serine, Thr-Threonine, Asp-Aspartic acid,Glu-Glutamic acid, Asn-Asparagine, Gln-Glutamine, Lys-Lysine,His-Histidine, Arg-Arginine, Phe-Phenylalanine, Tyr-Tyrosine,Trp-Tryptophan, Cys-Cysteine, Met-Methionine and Pro-Proline.

[0104] Polypeptides according to the invention may be used to raiseantibodies which will cross-react with prion proteins produced in a widerange of organisms. Our analyses have shown that since theconformational, topographic and electrostatic properties of polypeptidesaccording to the invention are such that they are highly likely toelicit the production of antibodies which will cross-react with prionproteins from several or many organisms, further advantages may arisefrom combining several variant polypeptides in a larger polypeptide.Such a polypeptide may have the general formula (IX):

[L _(a) −F] _(m) −[L _(b) −G] _(n) −L _(c)  (IX)

[0105] wherein F and G may each independently be a polypeptide orsub-fragment according to any one of Formulae I to VIIIb, L is a linkingsequence, a, b and c are each independently 0 or 1 and m and n are eachpositive numbers e.g. between 1 and 10 inclusive. L is preferably ashort, conformationally flexible section of polypeptide chain such as,for example and without limit (Seq. I.D. No: 38) Gly-Gly-Gly-Gly-Gly,(Seq. I.D. No: 39) Gly-Pro-Gly-Pro-Gly-Pro or (Seq. I.D. No: 40)Gly-Ser-Ala-Gly-Ser-Gly-Ala. It should be clear that each repeat mayoptionally have a different variant of a polypeptide according to theinvention.

[0106] It should be noted certain of the C-terminals correspond toN-terminals, particularly formula Va to formula Vb, formula Vc toformula I, formula I to formula II, formula II to formula III, formulaIII to formula VIIIa and formula VIIIb to formula IV. Advantage may betaken to this correspondence when producing larger polypeptidesaccording to formula IX. Linking sequences together with respective Xand Y moieties may be omitted and residues in brackets may be selectedso that either the regions of correspondence are duplicated or some orall of the duplicated residues are omitted. In the latter case it willbe seen that the C-terminal of one polypeptide merges with theN-terminal of the other polypeptide.

[0107] Polyvalent determinant analogues as defined by Formula IX may beeither what is referred to as pseudohomopolyvalent, wherein variants ofessentially the same determinant analogue are repeated in a singlepolypeptide chain and/or heteropolyvalent, wherein distinct determinantsare included in a single polypeptide. In addition, simple homopolyvalentpolypeptide immunogens, which contain multiple copies of the samevariant of one of the determinant analogues according to any one offormulae I to VIIIb, would also be expected to be effective, and arealso included within the scope of the present invention.

[0108] It is to be understood that any antigenically significantsubfragments and/or antigenically significant variants of theabove-identified polypeptide sequences which retain the general form andfunction of the parent polypeptide are included within the scope of thisinvention. In particular, the substitution of any of the specificresidues by residues having comparable conformational and/or physicalproperties, including substitution by rare (but naturally occurring,e.g. D-stereoisomers) or synthetic amino acid analogues, is included.For example, substitution of a residue by another in the same Set, asdefined below, is included within the ambit of the invention; Set 1-Ala,Val, Leu, Ile, Phe, Tyr, Trp and Met; Set 2-Ser, Thr, Asn and Gln; Set3-Asp and Glu; Set 4-Lys, His and Arg; Set 5-Asn and Asp; Set 6-Glu andGln; Set 7-Gly, Ala, Pro, Ser and Thr. D-stereoisomers of all amino acidtypes, may be substituted, for example, D-Phe, D-Tyr and D-Trp.

[0109] In preferred embodiments of the invention, X and Y if present mayindependently include one or more segments of protein sequence with theability to act as a T-cell epitope. For example, segments of amino acidsequence of the general formula 1-2-3-4, where 1 is Gly or a chargedamino acid (e.g. Lys, His, Arg, Asp or Glu), 2 is a hydrophobic aminoacid (e.g. Ile, Leu, Val, Met, Tyr, Phe, Trp, Ala), 3 is either ahydrophobic amino acid (as defined above) or an uncharged polar aminoacid (e.g. Asn, Ser, Thr, Pro, Gln, Gly), and 4 is a polar amino acid(e.g. Lys, Arg, His, Glu, Asp, Asn, Gin, Ser, Thr, Pro), appear to actas T-cell epitopes in at least some instances (Rothbard, J. B. & Taylor,W. R. (1988). A sequence pattern in common to T-cell epitopes. The EMBOJournal 7(1): 93-100). Similarly segments can be of the sequence1′-2′-3′-4′-5′, wherein 1′ is equivalent to 1 as defined earlier, 2′ to2, 3′ and 4′ to 3, and 5′ to 4 (ibid). Both forms are included withinthe scope of the present invention and one or more T-cell epitopes(preferably less than five) which may be of the type defined above ormay be of other structure and which may be separated by spacer segmentsof any length or composition, preferably less than five amino acidresidues in length and comprising for example residues selected fromGly, Ala, Pro, Asn, Thr, Ser or polyfunctional linkers such as non-aamino acids. It is possible for a C- or N-terminal linker to represent acomplete protein, thus obviating the possible need for conjugation to acarrier protein.

[0110] Also included within the scope of this invention are derivativesof the polypeptides according to any one formulae I to VIIIb in which Xor Y are or include a “retro-inverso” amino acid, i.e. a bifunctionalamine having a functional group corresponding to an amino acid. Forexample an analogue according to the invention and containing aretro-inverso amino acid may have the formula:

[0111] where R is any functional group, e.g. a glycine side chain, andA1 and A2 are preferably each a copy of one of the analogues definedherein (but not necessarily the same) attached by its—or C-terminal end.T-cell epitopes may optionally be included as discussed earlier.

[0112] Retro-inverso modification of peptides involves the reversal ofone or more peptide bonds to create analogues more resistant than theoriginal molecule to enzymatic degradation and offer one convenientroute to the generation of branched immunogens which contain a highconcentration of epitope for a medium to large immunogen. The use ofthese compounds in large-scale solution synthesis of retro-inversoanalogues of short-chain biologically active peptides has greatpotential.

[0113] Peptides according to the invention may be synthesized bystandard peptide synthesis techniques, for example using either standard9-fluorenylmethoxycarbonyl (F-Moc) chemistry (see, for example,Atherton, E. and Sheppard, R. C. (1985) J. Chem. Soc. Chem. Comm. 165)or standard butyloxycarbonate (T-Boc) chemistry although it is notedthat, more recently, the fluorenylmethoxycarbonyl (Fmoc)/tert-butylsystem, developed by Sheppard et al has found increasingly wideapplication (Sheppard, R. C. 1986 Science Tools, The LKB Journal 33, 9).The correctness of the structure and the level of purity, which willnormally be in excess of 85%, should be carefully checked, andparticular attention be given to the correctness of internal disulphidebridging arrangements when present. Various chromatographic analyses,including high performance liquid chromatography, and spectrographicanalyses, including Raman spectroscopy, may for example be employed forthis purpose.

[0114] It is to be understood that the polypeptides according to theinvention may be synthesized by any conventional method, either directlyusing manual or automated peptide synthesis techniques as mentionedabove, or indirectly by RNA or DNA synthesis and conventional techniquesof molecular biology and genetic engineering. Such techniques may beused to produce hybrid proteins containing one or more of thepolypeptides inserted into another polypeptide sequence.

[0115] Another aspect of the present invention therefore provides a DNAmolecule coding for at least one synthetic polypeptide according to theinvention, preferably incorporated into a suitable expression vectorreplicable in microorganisms or in mammalian cells. The DNA may also bepart of the DNA sequence for a longer product e.g. the polypeptides maybe expressed as parts of other proteins into which they have beeninserted by genetic engineering. One practical guide to such techniquesis “Molecular cloning: a laboratory manual” by Sambrook, J., Fritsch, E.F. and Maniatis, T. (2nd Edition, 1989).

[0116] It should be noted that analogues incorporating retro-inversoamino acid derivatives cannot be made directly using a recombinant DNAsystem. However, the basic analogues can, and they can then be purifiedand chemically linked to the retro-inverso amino-acids using standardpeptide/organic chemistry. A practical and convenient novel procedurefor the solid-phase synthesis on polyamide-type resin of retro-inversopeptides has been described recently [Gazerro, H., Pinori, M. & Verdini,A. S. (1990). A new general procedure for the solid-phase synthesis ofretro-inverso peptides. In “Innovation and Perspectives in Solid PhaseSynthesis” Ed. Roger Epton. SPCC (UK) Ltd, Birmingham, UK].

[0117] The polypeptides are optionally linked to a carrier molecule,either through chemical groups within the polypeptides themselves orthrough additional amino acids added at either the C- or N-terminus, andwhich may be separated from the polypeptides themselves or surrounded byone or more additional amino acids, in order to render them optimal fortheir immunological function. Many linkages are suitable and include forexample use of the side chains of Tyr, Cys and Lys residues. Suitablecarriers include, for example, purified protein derivative of tuberculin(PPD), tetanus toxoid (TT), cholera toxin and its B subunit, ovalbumin,bovine serum albumin (BSA), soybean trypsin inhibitor (STI), muramyldipeptide (MDP) and analogues thereof, diphtheria toxoid (DPT), keyholelimpet haemocyanin (KLH) and Braun's lipoprotein although other suitablecarriers will be readily apparent to the skilled person. For example,multiple antigen peptides may be used such as those comprising apolylysyl core, e.g. heptalysyl, bearing reactive amine termini.Polypeptide antigens according to the invention may be reacted with, orsynthesized on, the amino termini and different polypeptide antigens maybe reacted with the same core or carrier. When using PPD as a carrierfor polypeptides according to the invention, a higher titre ofantibodies is achieved if the recipient of the polypeptide-PPD conjugateis already tuberculin sensitive, e.g. by virtue of earlier BCGvaccination. In the case of a human vaccine it is worth noting that inthe UK and many other countries the population is routinely offered BCGvaccination and is therefore largely PPD-sensitive. Hence PPD isexpected to be a preferred carrier for use in such countries.

[0118] The mode of coupling the polypeptide to the carrier will dependon the nature of the materials to be coupled. For example, a lysineresidue in the carrier may be coupled to a C-terminal or other cysteineresidue in a polypeptide by treatment withN-γ-maleimidobutyryloxy-succinimide (Kitagawa, T. & Ackawa, T. (1976) J.Biochem. 79, 233). Alternatively, a lysine residue in the carrier may beconjugated to a glutamic or aspartic acid residue in the peptide usingisobutylchloroformate (Thorell, J. I. De Larson, S. M. (1978)Radioimmunoassay and related techniques: Methodology and clinicalapplications, p.288). Other coupling reactions and reagents have beendescribed in the literature.

[0119] The polypeptides, either alone or linked to a carrier molecule,may be administered by any route (e.g. parenteral, nasal, oral, rectal,intra-vaginal), with or without the use of conventional adjuvants (suchas aluminum hydroxide or Freund's complete or incomplete adjuvants)and/or other immunopotentiating agents. The invention also includesformulation of polypeptides according to the invention in slow-releaseforms, such as a sub-dermal implant or depot comprising, for example,liposomes (Allison, A. C. & Gregoriadis, G. (1974) Nature (London) 252,252) or biodegradable microcapsules manufactured from co-polymers oflactic acid and glycolic acids (Gresser, J. D. and Sanderson, J. E.(1984) in “Biopolymer Controlled Release Systems” pp 127-138, Ed. D. L.Wise).

[0120] Polypeptides according to the invention may be used either aloneor linked to an appropriate carrier, as:

[0121] (a) As ligands in assays of, for example, serum from patients oranimals;

[0122] (b) Peptide vaccines, for use in prophylaxis,

[0123] (c) As quality control agents in testing, for example, bindinglevels of antibodies raised against the polypeptides;

[0124] (d) As antigenic agents for the generation of monoclonal orpolyclonal antibodies by immunization of an appropriate animal, suchantibodies being of use for (i) the scientific study of prion proteins,(ii) as diagnostic agents, e.g. as part of immunohistochemical reagents,(iii) for the passive immunisation of animals or patients, either as atreatment for encephalophathies or in combination with other agents,(iv) as a means of targeting other agents to regions comprising prionproteins, such agents either being linked covalently or otherwiseassociated, e.g. as in liposomes containing such agents andincorporating antibodies raised against any of the antigenicpolypeptides and (v) for use as immunogens to raise anti-idiotypeantibodies; such anti-idiotype antibodies also form part of thisinvention. The invention further provides for genetically engineeredforms or sub-components, especially V_(H) regions, of antibodies raisedagainst the polypeptides, and of ovinised, bovinised, or humanized formsof antibodies initially raised against the polypeptides in otheranimals, using techniques described in the literature; and

[0125] (e) The treatment of encephalopathies, either by displacing thebinding of prion proteins to human or animal cells or by disturbing thethree-dimensional organization of the protein in vivo; as well as aidingthe scientific study of prion proteins in vitro.

[0126] In respect of detection and diagnosis, of prion proteins orantibodies against prion proteins, the skilled person will be aware of avariety of immunoassay techniques known in the art, inter alia, sandwichassay, competitive and non-competitive assays and the use of direct andindirect labeling.

[0127] A further aspect of the invention provides a kit for detectingprion proteins or antibodies against prion proteins which comprises atleast one synthetic polypeptide according to the invention.

[0128] The preparation of polyclonal or monoclonal antibodies, humanizedforms of such antibodies (see, for example, Thompson K. M. et al (1986)Immunology 58, 157-160), single domain antibodies (see, for example,Ward, E. S., Gussow, D., Griffiths, A. D., Jones, P. and Winter, G.(1989) Nature 341, 544-546), and antibodies which might cross theblood-brain barrier, which bind specifically to a synthetic polypeptideaccording to the present invention, may be carried out by conventionalmeans and such antibodies are considered to form part of this invention.Antibodies according to the invention are, inter alia, of use in amethod of diagnosing mammalian encephalopathies which comprisesincubating a sample of tissue or body fluid of mammal with an amount ofantibody as described herein and determining whether, and if desired theextent to which and/or rate at which, cross-reaction between said sampleand said antibody occurs. Diagnostic kits which contain at least one ofsaid antibodies also form part of this invention.

[0129] A further aspect of the invention provides synthetic polypeptidesfor use in therapy or prophylaxis of mammalian encephalopathies and/orstimulating the mammalian immune system and/or blocking the cellularbinding or aggregation of the prion proteins and for the preparation ofmedicaments suitable for such uses. Also included are pharmaceuticalcompositions containing, as active ingredient, at least one polypeptideor polypeptide-carrier conjugate as described herein in association withone or more pharmaceutically acceptable adjuvants, carriers and/orexcipients. The compositions may be formulated for oral, rectal, nasalor especially parenteral administration (including intra-CNSadministration).

[0130] The invention further provides a method of therapy or prophylaxisof mammalian encephalopathies and/or of stimulating the mammalian immunesystem and/or of blocking the cellular binding or aggregation of theprion proteins, which comprises administering an amount of a polypeptideas hereinbefore defined, either in isolation or in combination withother agents for the treatment of encephalopathies.

[0131] Discrimination between natural PrP^(C) and PrP^(SC) is highlydesired since PrP^(C) is found in normal subjects and both PrP^(C) andPrP^(SC) are found in a diseased subject. We have found that peptidesequences according to the invention, preferably those relating toregions A, B and C, and significant sub-fragments thereof may be used todiscriminate between natural PrP^(C) and infective PrP^(SC). Also,antibodies raised against these peptide sequences and sub-fragments andthe nucleotide sequences which code for such peptide sequences andsub-fragments may also be used to discriminate between PrP^(C) andPrP^(SC). Accordingly, the invention provides a method of discriminatingbetween PrP^(C) and PrP^(SC) in which a sample is contacted with asubstance selected from peptide sequences according to the invention,preferably those relating to regions A, B and C, and significantsub-fragments thereof, antibodies raised against said sequences andsub-fragments and the presence or absence of PrP^(SC) is determined.

[0132] In some instances discrimination may be enhanced by pretreatmentof the sample, for example by pre-digestion with enzymes e.g. proteinaseK, or denaturation by strong alkali e.g. 6M guanidine hydrochloride orby a combination of such treatments.

[0133] It will be preferable to use the peptide sequences, antibodiesand nucleotide sequences which relate to the specific subject undertest, e.g. bovine sequences and antibodies for cattle, ovine sequencesand antibodies for sheep.

[0134] It may be advantageous to immunize with a cocktail containing (i)a given analogue conjugated to more than one type of carrier molecule,and/or (ii) more than one kind of analogue conjugated to the samecarrier molecule. Moreover, any of the peptide analogues, theirconjugates, and cocktails thereof may be administered in any suitableadjuvant or delivery system, and more than one adjuvant or deliverysystem may be combined to form a so-called “super-cocktail”. Preferredadjuvants and delivery systems include aluminum hydroxide (alum),liposomes, micelles, niosomes, ISCOMS, Brauns lipoprotein and whole-cellor components of microbial animal vaccines.

EXAMPLE 1

[0135] A preferred bovine form of formula II (Seq. I.D. No: 41)Ala-Met-Ser-Arg-Pro-Leu-Ile-His-Phe-Gly-Ser-Asp-Tyr-Glu-Asp-Arg-Tyr-Tyr-Arg-Glu-Asn-Met-His-Arg-Gly-Cys (related to Seq. I.D. No: 7) in which the C-terminal Y extension isGly-Cys according to the invention is synthesised using standardsolid-phase Fmoc methodologies. The peptide is cleaved from the resin inthe presence of trifluroacetic acid and subsequent purification isachieved by gel filtration, ion exchange chromatography and reversephase high performance liquid chromatography. The peptide is conjugatedto a variety of carriers by MBS (m-Maleimido-benzoyl-N-hydroxysuccinimide ester), a well-known hetero-bifunctional reagent.

[0136] Examples of carriers include KLH, BSA and TT which have beenshown to provide necessary immunopotentiating properties to B cellepitopes.

[0137] The peptide carrier conjugates are emulsified in Freund'sComplete Adjuvant and are administered intramuscularly to mice.Subsequent booster injections are given in Freund's Incomplete Adjuvant.

[0138] The ensuing serum antibody response is monitored throughout theimmunization schedule by enzyme immunoassay (ELISA) using immobilizedantigen (formula II), coupled to BSA, the serum sample under test, andan enzyme-labeled anti-mouse antibody.

[0139] In this example, use of carriers, adjuvants and delivery systemsand booster injections are effected in order to determine an optimalprotocol for producing anti-formula II antibodies.

EXAMPLE 2

[0140] Antibodies to formula II are used as diagnostic reagents forassaying the presence of prion protein in serum, in “cell carriers” inserum and in tissue biopsies of injected animal species.

[0141] A direct enzyme immunoassay (ELISA) can detect the presence ofextracted prion protein by its immobilization onto a solid substrate.Reaction of mouse antisera raised to formula II with native prionprotein is detected with an enzyme-labeled anti-mouse antiserum. Thereaction is quantified by addition of a suitable substrate and readingthe optical density of the color produced.

[0142] Furthermore, immunohistochemical diagnosis of prion proteins intissue biopsies can be performed by reacting anti-formula II antibodieswith paraffin wax embedded or frozen tissue. Reactions can be detectedusing a standard indirect immunoperoxidase technique.

EXAMPLE 3

[0143] Materials and Methods

[0144] Peptide Synthesis

[0145] The following peptides were synthesised using standardsolid-phase Fmoc methodologies. Peptide II: (Seq. I.D. No:42)Ser-Ala-Met-Ser-Arg-Pro-Leu-Ile-His-Phe-Gly-Asn-Asp-Tyr-Glu-Asp-Arg-Tyr-Tyr-Gly-Cys

[0146] (A preferred ovine sub-fragment of formula II). Peptide BII:(Seq. I.D. No:43) Ser-Ala-Met-Ser-Arg-Pro-Leu-Ile-His-Phe-Gly-Ser-Asp-Tyr-Glu-Asp-Arg-Tyr-Tyr-Gly-Cys

[0147] (A preferred bovine sub-fragment of formula II). Peptide III:(Seq. I.D. No:44) Asn-Met-Tyr-Arg-Tyr-Pro-Asn-Gln-Val-Tyr-Tyr-Arg-Pro-Val-Asp-Arg-Tyr-Ser-Asn-Gln-Asn-Asn-Phe-Val- His-Gly-Cys

[0148] (A preferred ovine sequence of formula III (p8, in 30-32).Peptide BIII: (Seq. I.D. No:45)Asn-Met-His-Arg-Tyr-Pro-Asn-Gln-Val-Tyr-Tyr-Arg-Pro-Val-Asp-Gln-Tyr-Ser-Asn-Gln-Asn-Asn-Phe-Val- His-Gly-Cys

[0149] (A preferred bovine sequence of formula III (p8, in 26-28).Peptide Vb: (Seq. I.D. No:46)Gly-Gln-Pro-His-Gly-Gly-Gly-Trp-Gly-Gln-Pro-His-Gly-Gly-Gly-Trp-Gly-Gln-Pro-His-Gly-Gly-Gly-Trp- Gly-Cys

[0150] (A preferred ovine/bovine sequence of formula Vb). Peptide Vc:(Seq. I.D. No:47) Gly-Gln-Gly-Gly-Ser-His-Ser-Gln-Trp-Asn-Lys-Pro-Ser-Lys-Pro-Lys-Thr-Asn-Met-Lys-His-Val-Gly-Cys

[0151] (A preferred ovine sequence of formula Vc). Peptide VIIIb:Cys-Ile-Thr-Gln-Tyr-Gln-Arg-Glu-Ser-Gln-Ala-Tyr-Tyr-Gln-Arg (Seq. I.D.No:48)

[0152] (A preferred ovine/bovine sequence of formula VIIIb). Peptide Va:(Seq. I.D. No:49) Gly-Gly-Trp-Asn-Thr-Gly-Gly-Ser-Arg-Tyr-Pro-Gly-Gln-Gly-Ser-Pro-Gly-Gly-Asn-Arg-Tyr-Pro-Pro-Gln- Gly-Gly-Gly-Cys PeptideVIIIa: (Seq. I.D. No:50)Val-Asn-Ile-Thr-Val-Lys-Gln-His-Thr-Val-Thr-Thr-Thr-Thr-Lys-Gly-Glu-Asn-Phe-Thr-Glu-Gly-Cys

[0153] (A preferred ovine sequence of formula VIIIa). Peptide I: (Seq.I.D. No:51) Lys-His-Met-Ala-Gly-Ala-Ala-Ala-Ala-Gly-Ala-Val-Val-Gly-Gly-Leu-Gly-Gly-Tyr-Met-Leu-Gly-Ser-Ala- Met-Ser-Arg-Gly-Cys.

[0154] Peptides I, II, BII, III, BIII, Va, Vb, Vc and VIIIa weresynthesised with the C-terminal extension according to the invention.The peptides were cleaved from the resin in the presence oftrifluoroacetic acid and subsequent purification was achieved by reversephase high performance liquid chromatography. All peptides had a purityof 85% or more.

[0155] Conjugation of Peptides to Ovalbumin

[0156] Peptides were conjugated through their C-terminal (peptides II,BII, III, BIII, Vb and Vc) or N-terminal (peptide VIIIb) Cys residues.Peptides were dissolved in dimethyl sulphoxide (DMSO) to a concentrationof 10 mg/ml. Preactivated ovalbumin (Pierce, Inject Kit) was resuspendedin 1 ml of distilled water, and equal volumes of preactivated ovalbuminand peptide were mixed and the solution allowed to stand at roomtemperature for 3 hours. The conjugate was dialyzed overnight againstphosphate buffered saline (PBS) to remove the DMSO and unconjugatedpeptide.

[0157] The extent of conjugation was determined by measuring thefree-thiol content using an Ellman's assay and by monitoring theincrease in the molecular mass of the conjugate by SDS-PAGE (sodiumdodecyl sulphate-polyacrylamide gel electrophoresis).

[0158] Generation of Rabbit Antisera.

[0159] Antiserum was raised against each of the peptide conjugates intwo female New Zealand White rabbits. Each rabbit received an amount ofconjugate equivalent to 40 μg of peptide for both the primaryinoculation and the boosters. Rabbits were injected as follows: Day 0:Conjugate in Freund's Complete Adjuvant (1:1, v/v) intramuscularly. Day21: Conjugate in Freund's Incomplete Adjuvant (1:1, v/v)intramuscularly. Day 31: Conjugate on its own intraperitoneally.

[0160] Animals were bled on day 41, and the sera assayed foranti-peptide antibody by ELISA (using free peptide as the coatingantigen). The sera were also used in immunoblot and dot blot assays tosee if they could recognize proteins from the brain homogenates.

[0161] Preparation of Brain Homogenates

[0162] Scrapie-free brain material was obtained from a flock of NewZealand sheep in quarantine.

[0163] Scrapie-infected brain material was obtained from a Department ofAgriculture and had been histopathalogically diagnosed as being scrapieinfected.

[0164] BSE-infected brain material was obtained via a governmentAgriculture Department and had been histopathalogically certified asbeing BSE infected.

[0165] BSE-free material was obtained through a private source.

[0166] Ha27-30 is brain material obtained from an inbred hamster scrapiemodel, which has been shown to contain a high level of thescrapie-infective agent. It was used as a positive control.

[0167] Small samples of infected and uninfected brain were weighed and10% (w/v) homogenates made up in 10% (v/v) solution of Sarkosyl in 25 mMTris-HCl pH 7.4 (homogenization buffer). The homogenate was incubated at4° C. for 30 mins and then spun at 6000×g for 30 mins. The supernatantwas collected and the protein content determined using the BCA proteinassay kit (Pierce). The protein concentration was adjusted to 3 mg/mlusing homogenizaton buffer.

[0168] ELISA (Enzyme-Linked Immunosorbent Assay)

[0169] A 8 μM solution of free peptide in PBS was used as the coatingantigen. Microtitre plates were coated by adding 50 μl of the antigenconcentration to each well and then incubated for 1 hour at 37° C. toallow binding to occur. Each well was washed times, for 2 minutes, with300 μl of PBS containing 0.05% Tween 20. After washing, the plates wereblocked by incubating for 1 hour at 37° C. with PBS containing 0.3%Tween 20 and 3% non-fat milk. An aliquot of 50 μl of primary antibody(i.e. antisera) diluted in PBS was added to the appropriate wells andthe plates incubated for 1 hour at 37° C. Plates were washed as before,and then incubated with Horseradish peroxidase conjugated swineanti-rabbit immunoglobulin (anti Ig/HRP) at a dilution of 1:1000 in PBSfor 1 hour at 37° C. The plates were washed and 50 μl of OPD(O-phenylenediamine dihydrochloride substrate (10 mg/ml) in citratebuffer) added to each well and the reaction allowed to proceed at roomtemperature for 10 minutes, before being stopped by the addition ofsulphuric acid. The absorbence of each well was measured at 492 nm usingan ELISA plate reader. The titres were recorded as the dilutions whichgave a positive optical density (OD) reading at least 3 times that ofthe background. The background was taken as the OD readings from wellswhich had not been coated with antigen.

[0170] Dot Blot Detection of PrP in Brain Homogenates

[0171] The brain homogenates prepared as described earlier were diluted10-fold in PBS, and 100 μl of homogenates (containing 30 μg totalprotein) were applied to nitrocellulose filters using BRL 96 well vacuummanifold. The filters were dried for 1 hour at room temperature. Thefilters were then either wet with TBST (10 mM Tris-HCl pH 7.4, 150 mMNaCl, 0.05% Tween 20) and PrP detected as described in the immunoblots,or the protein sample further treated. This further treatment of thesample included digestion of the protein on the filter using 100 μg/mlproteinase K in TBST for 90 minutes at room temperature. The proteinaseK was inactivated by the addition of PMSF (phenylmethylsulphonylfluoride) to a concentration of 5 mM in TBST. After protein digestion,some samples were also denatured by incubating the filters in 6Mguanidine HCl containing 5 mM PMSF for 10 minutes. The guanidine wasremoved by 3 washes with TBST prior to incubation with the primaryantibody.

[0172] Immunoblots. (Western Blots)

[0173] SDS-PAGE was performed on the brain homogenates, prepared asdescribed previously, using standard techniques. The samples within thegel were transferred onto nitrocellulose in a Biorad transblot usingTowbin Buffer (25 mM Tris, 190 mM glycine and 0.1% SDS) at 70 mAovernight. The nitrocellulose filter was blocked with 5% non-fat milkfor 30 minutes at room temperature. The primary antibody (i.e. antisera)diluted in TBST was applied for 3 hours at room temperature, the filterwashed 3 times for 10 minutes in TBST and the filter incubated for 2hours at room temperature with the alkaline phosphatase-conjugated swineanti-rabbit immunoglobulin diluted at a dilution of 1:2000. Afterwashing, the protein bands were detected using the NBT/BCIP (nitro-bluetetrazolium; 5-bromo-4-chloro-3-indolyl phosphate) substrate (BoehringerMannheim).

[0174] Results

[0175] 1) Antibody titres: Good antibody titres to the peptides wereobtained in all cases, though the level varied enormously. The peptidewhich gave the highest titre, also gave the best results in the dotblots.

[0176] 2) Dot Blot Data: Uninfected tissue would be expected to containonly normal prion protein (PrP^(C)). Infected tissue would be expectedto contain both the normal and the diseased (PrP^(SC)) forms of PrP.

[0177] PrP^(SC) has a molecular weight of approximately 33-35 kD.PrP^(SC) has a molecular weight of approximately 27-30 kD and is missingan N-terminal segment that is present in the PrP^(C) form. Otherwise,the amino acid sequence of PrP^(SC) is exactly the same as that ofPrP^(C). Probably the most significant characteristic of (PrP^(SC) isresistance to enzyme degradation with proteinase K, a non-specificprotein-digesting enzyme.

[0178] When a protein sample is treated with proteinase K any PrP^(C)should be completely digested. Therefore, in a sample containing onlyPrP^(C), no PrP of any form will remain after proteinase K treatment.However, in a sample containing PrP^(C) and PrP^(SC) (i.e. a diseasedsample), PrP^(SC) will remain after treatment.

[0179] There are antibodies currently available which recognizePrP^(SC), but they only recognize the denatured protein. Therefore afterproteinase K treatment, samples in the dot blot test were treated withguanidine HCl, a denaturing agent, so that such antibodies could be usedto detect PrP^(SC).

[0180] The data are given in Tables I-V.

[0181] Peptide II

[0182] Good titres. Dot blots appear to indicate that somediscrimination is occurring. Negative results were obtained from theWestern blots.

[0183] Peptide III

[0184] Reasonable titres. Possibly there is recognition of anon-specific (perhaps non-protein) component in the proteinase K andguanidine treated samples. Negative results were obtained from theWestern blots.

[0185] Peptide Vb

[0186] Good titres. Although it appears that there might be somediscrimination occurring, the Vb peptide in fact occurs within theN-terminal region that is missing in PrP^(SC). One would therefore notexpect to see any recognition in the infected material treated withproteinase K and guanidine. However, one possible explanation is thatthe PrP^(C) present in the infected

[0187] material has not been completely digested by the proteinase K.Negative results were obtained from the Western blots.

[0188] Peptide Vc: Excellent titres. These results are exactly asexpected. As mentioned previously, antibodies which recognize PrP^(SC)generally only recognize the protein in its denatured state. Infectedand uninfected samples, as well as containing PrP^(SC) and/or PrP^(C) intheir “native” states, will also contain both PrP forms in variousstages of denaturation due to natural protein turnover within cells. Forthis reason, antibodies would be expected to detect all three untreatedsamples. However, proteinase K treatment will digest PrP^(C) and anypartially denatured PrP^(SC) leading to a loss of antibody recognitionin all samples (assuming the antibody only recognizes denatured PrP).The addition of guanidine should restore antibody recognition inmaterial which had originally contained PrP^(SC). Western blots showedup the expected protein bands at the correct molecular weights.

[0189] Peptide VIIIb:

[0190] Reasonable titre. There may be recognition of a non-specificcomponent. Negative results were obtained form the Western blots.

[0191] Peptides BII & BIII:

[0192] The titres are reasonable and there are strong positive resultsfrom untreated normal and infected bovine brain material.

[0193] In summary, good anti-peptide titres obtained in all cases, theWestern blots only worked well in the case of peptide Vc, which alsogave the highest titre and the dot blots show that there is somediscrimination occurring between PrP^(C) and PrP^(SC) with peptide Vc.Data from peptide II also suggests that discrimination is occurring.TABLE I Results from ovine peptide sequences Dot Blot Pept/ Anti- OvineProt carrier body Brain Prot K + West Ratio number Titre Material UntrtK Gua Blot II 8:1 93 20,000 infected ++ + + normal ++ − − Ha27-30 +/−+/− +/− II 8:1 94 20,000 infected ++ + + normal ++ − − Ha27-30 + + + III6:1 101 5,000 infected ++ + + normal ++ − + Ha27-30 ++ − ++ III 6:1 1025,000 infected +++ + + normal +++ +/− +/− Ha27-30 +++ ++ ++ Vc 5:1 97160,000 infected +++ +/− +++ + normal +++ +/− +/− + Ha27-30 +++ ++ +++ +Vc 5:1 98 320,000 infected +++ +/− +++ + normal +++ +/− +/− + Ha27-30+++ +/− +++ +

[0194] TABLE II Results from ovine peptide sequences Dot Blot Pept/Anti- Bovine Prot carrier body Brain Prot K + West Ratio number TitreMaterial Untrt K Gua Blot II 8:1 93 20,000 infected ++ + + normal ++ + +Ha27-30 +/− +/− +/− II 8:1 94 20,000 infected ++ + + normal ++ + +Ha27-30 + + + III 6:1 101 5,000 infected + + + + normal + + + + Ha27-30++ ++ ++ III 6:1 102 5,000 infected ++ + ++ normal ++ + + Ha27-30 ++ ++++ Vc 5:1 97 160,000 infected +++ + ++ normal ++ + + Ha27-30 +++ ++ +++Vc 5:1 98 320,000 infected +++ + ++ normal ++ +/− +/− Ha27-30 +++ +/31+++

[0195] TABLE III Results from ovine/bovine peptide sequences Anti- DotBlot Pept/ body Ovine Prot carrier num- Brain Prot K + West Ratio berTitre Material Untrt K Gua Blot Vb 6:1 95 50,000 infected ++ + + normal++ − − Ha27-30 ++ ++ ++ Vb 6:1 96 10,000 infected ++ + + normal ++ − −Ha27-30 ++ ++ ++ VIIIb 12:1 103 3,000 infected ++ + + normal ++ + +Ha27-30 ++ +/− +/− VIIIb 12:1 104 3,000 infected + + + normal + + +Ha27-30 ++ + +

[0196] TABLE III Results from ovine/bovine peptide sequences Anti- DotBlot Pept/ body Bovine Prot carrier num- Brain Prot K + West Ratio berTitre Material Untrt K Gua Blot Vb 6:1 95 50,000 infected ++ + + normal++ + + Ha27-30 ++ ++ ++ Vb 6:1 96 10,000 infected ++ + + normal ++ + +Ha27-30 ++ ++ ++ VIIIb 12:1 103 3,000 infected ++ + + normal ++ + +Ha27-30 ++ +/− +/− VIIIb 12:1 104 3,000 infected + + + normal ++ +/− +/−Ha27-30 + + +

[0197] TABLE V Results from bovine peptide sequences Anti- Dot BlotPept/ body Bovine Prot carrier num- Brain Prot K + West Ratio ber TitreMaterial Untrt K Gua Blot BII 9:1 105 100,000 infected +++ + + normal+++ + + Ha27-30 + + + BII 9:1 106 100,000 infected +++ + + normal+++ + + Ha27-30 + + + BIII 5:1 107 20,000 infected +++ +/− +/− normal+++ +/− +/− Ha27-30 + + + BIII 5:1 108 10,000 infected +++ +/− +/−normal +++ +/− +/− Ha27-30 + + +

[0198]

1 67 33 amino acids amino acid single linear peptide Modified-site 1/label= X /note= “X may be absent or present independently of Y anddenotes one or more amino acid(s)” Modified-site 33 /label= Y /note= “Ymay be absent or present independently of X and denotes one or moreamino acid(s) 1 Xaa Met Lys His Val Ala Gly Ala Ala Ala Ala Gly Ala ValVal Gly 1 5 10 15 Gly Leu Gly Gly Tyr Met Leu Gly Ser Ala Met Ser ArgPro Leu Ile 20 25 30 Xaa 33 amino acids amino acid single linear peptideModified-site 1 /label= X /note= ”X may be absent or presentindependently of Y and denotes one or more amino acid(s)“ Modified-site33 /label= Y /note= ”Y may be absent or present independently of X anddenotes one or more amino acid(s)“ 2 Xaa Met Lys His Met Ala Gly Ala AlaAla Ala Gly Ala Val Val Gly 1 5 10 15 Gly Leu Gly Gly Tyr Met Leu GlySer Ala Met Ser Arg Pro Ile Ile 20 25 30 Xaa 19 amino acids amino acidlinear peptide Modified-site 1 /label= X /note= ”X may be absent orpresent independently of Y and denotes one or more amino acid(s)“Modified-site 19 /label= Y /note= ”Y may be absent or presentindependently of X and denotes one or more amino acid(s)“ 3 Xaa His ValAla Gly Ala Ala Ala Ala Gly Ala Val Val Gly Gly Leu 1 5 10 15 Gly GlyXaa 19 amino acids amino acid linear peptide Modified-site 1 /label= X/note= ”Y may be absent or present independently of X and denotes one ormore amino acid(s)“ Modified-site 19 /label= Y /note= ”Y may be absentor present independently of X and denotes one or more amino acid(s)“ 4Xaa Gly Gly Leu Gly Gly Tyr Met Leu Gly Ser Ala Met Ser Arg Pro 1 5 1015 Leu Ile Xaa 19 amino acids amino acid single linear peptideModified-site 1 /label= X /note= ”X may be absent or presentindependently X of Y and denotes one or more amino acid(s)“Modified-site 19 /label= Y /note= ”Y may be absent or presentindependently of X and denotes one or more amino acid(s)“ 5 Xaa His MetAla Gly Ala Ala Ala Ala Gly Ala Val Val Gly Gly Leu 1 5 10 15 Gly GlyXaa 19 amino acids amino acid single linear peptide Modified-site 1/label= X /note= ”X may be absent or present independently of Y anddenotes one or more amino acid(s)“ Modified-site 19 /label= Y /note= ”Ymay be absent or present independently of X and denotes one or moreamino acid(s)“ 6 Xaa Gly Gly Leu Gly Gly Tyr Met Leu Gly Ser Ala Met SerArg Pro 1 5 10 15 Ile Ile Xaa 31 amino acids amino acid single linearpeptide Modified-site 1 /label= X /note= ”X may be absent or presentindependently of Y and denotes one or more amino acid(s)“ Modified-site31 /label= Y /note= ”Y may be absent or present independently of X anddenotes one or more amino acid(s)“ 7 Xaa Ser Ala Met Ser Arg Pro Leu IleHis Phe Gly Ser Asp Tyr Glu 1 5 10 15 Asp Arg Tyr Tyr Arg Glu Asn MetHis Arg Tyr Pro Asn Gln Xaa 20 25 30 31 amino acids amino acid singlelinear peptide Modified-site 1 /label= X /note= ”X may be absent orpresent independently of Y and denotes one or more amino acid(s)“Modified-site 31 /label= Y /note= ”Y may be absent or presentindependently of X and denotes one or more amino acid(s)“ 8 Xaa Ser AlaMet Ser Arg Pro Leu Ile His Phe Gly Asn Asp Tyr Glu 1 5 10 15 Asp ArgTyr Tyr Arg Glu Asn Met Tyr Arg Tyr Pro Asn Gln Xaa 20 25 30 31 aminoacids amino acid single linear peptide Modified-site 1 /label= X /note=”X may be absent or present independently of Y and denotes one or moreamino acid(s)“ Modified-site 31 /label= Y /note= ”Y may be absent orpresent independently of X and denotes one or more amino acid(s)“ 9 XaaSer Ala Met Ser Arg Pro Ile Ile His Phe Gly Ser Asp Tyr Glu 1 5 10 15Asp Arg Tyr Tyr Arg Glu Asn Met His Arg Tyr Pro Asn Gln Xaa 20 25 30 25amino acids amino acid single linear peptide Modified-site 1 /label= X/note= ”X may be absent or present independently of Y and denotes one ormore amino acid(s)“ Modified-site 25 /label= Y /note= ”Y may be absentor present independently of X and denotes one or more amino acid(s)“ 10Xaa Ser Ala Met Ser Arg Pro Leu Ile His Phe Gly Ser Asp Tyr Glu 1 5 1015 Asp Arg Tyr Tyr Arg Glu Asn Met Xaa 20 25 25 amino acids amino acidsingle linear peptide Modified-site 1 /label= X /note= ”X may be absentor present independently of Y and denotes one or more amino acid(s)“Modified-site 25 /label= Y /note= ”Y may be absent or presentindependently of X and denotes one or more amino acid(s)“ 11 Xaa Ser AlaMet Ser Arg Pro Leu Ile His Phe Gly Asn Asp Tyr Glu 1 5 10 15 Asp ArgTyr Tyr Arg Glu Asn Met Xaa 20 25 25 amino acids amino acid singlelinear peptide Modified-site 1 /label= X /note= ”X may be absent orpresent independently of Y and denotes one or more amino acid(s)“Modified-site 25 /label= Y /note= ”Y may be absent or presentindependently of X and denotes one or more amino acid(s)“ 12 Xaa Ser AlaMet Ser Arg Pro Ile Ile His Phe Gly Ser Asp Tyr Glu 1 5 10 15 Asp ArgTyr Tyr Arg Glu Asn Met Xaa 20 25 31 amino acids amino acid singlelinear peptide Modified-site 1 /label= X /note= ”X may be absent orpresent independently of Y and denotes one or more amino acid(s)“Modified-site 31 /label= Y /note= ”Y may be absent or presentindependently of X and denotes one or more amino acid(s)“ 13 Xaa Asn MetHis Arg Tyr Pro Asn Gln Val Tyr Tyr Arg Pro Val Asp 1 5 10 15 Gln TyrSer Asn Gln Asn Asn Phe Val His Asp Cys Val Asn Xaa 20 25 30 31 aminoacids amino acid single linear peptide Modified-site 1 /label= X /note=”X may be absent or present independently of Y and denotes one or moreamino acid(s)“ Modified-site 31 /label= Y /note= ”Y may be absent orpresent independently of X and denotes one or more amino acid(s)“ 14 XaaAsn Met Tyr Arg Tyr Pro Asn Gln Val Tyr Tyr Arg Pro Val Asp 1 5 10 15Arg Tyr Ser Asn Gln Asn Asn Phe Val His Asp Cys Val Asn Xaa 20 25 30 31amino acids amino acid single linear peptide Modified-site 1 /label= X/note= ”X may be absent or present independently of Y and denotes one ormore amino acid(s)“ Modified-site 31 /label= Y /note= ”Y may be absentor present independently of X and denotes one or more amino acid(s)“ 15Xaa Asn Met His Arg Tyr Pro Asn Gln Val Tyr Tyr Arg Pro Met Asp 1 5 1015 Glu Tyr Ser Asn Gln Asn Asn Phe Val His Asp Cys Val Asn Xaa 20 25 3028 amino acids amino acid single linear peptide Modified-site 1 /label=X /note= ”X may be absent or present independently of Y and denotes oneor more amino acid(s)“ Modified-site 28 /label= Y /note= ”Y may beabsent or present independently of X and denotes one or more aminoacid(s)“ 16 Xaa Arg Tyr Pro Asn Gln Val Tyr Tyr Arg Pro Val Asp Gln TyrSer 1 5 10 15 Asn Gln Asn Asn Phe Val His Asp Cys Val Asn Xaa 20 25 28amino acids amino acid single linear peptide Modified-site 1 /label= X/note= ”X may be absent or present independently of Y and denotes one ormore amino acid(s)“ Modified-site 28 /label= Y /note= ”Y may be absentor present independently of X and denotes one or more amino acid(s) “ 17Xaa Arg Tyr Pro Asn Gln Val Tyr Tyr Arg Pro Val Asp Arg Tyr Ser 1 5 1015 Asn Gln Asn Asn Phe Val His Asp Cys Val Asn Xaa 20 25 28 amino acidsamino acid single linear peptide Modified-site 1 /label= X /note= ”X maybe absent or present independently of Y and denotes one or more aminoacid(s)“ Modified-site 28 /label= Y /note= ”Y may be absent or presentindependently of X and denotes one or more amino acid(s)“ 18 Xaa Arg TyrPro Asn Gln Val Tyr Tyr Arg Pro Met Asp Glu Tyr Ser 1 5 10 15 Asn GlnAsn Asn Phe Val His Asp Cys Val Asn Xaa 20 25 31 amino acids amino acidsingle linear peptide Modified-site 1 /label= X /note= ”X may be absentor present independently of Y and denotes one or more amino acid(s)“Modified-site 31 /label= Y /note= ”Y may be absent or presentindependently of X and denotes one or more amino acid(s)“ 19 Xaa Tyr TyrGln Arg Gly Ala Ser Val Ile Leu Phe Ser Ser Pro Pro 1 5 10 15 Val IleLeu Leu Ile Ser Phe Leu Ile Phe Leu Ile Val Gly Xaa 20 25 30 31 aminoacids amino acid single linear peptide Modified-site 1 /label= X /note=”X may be absent or present independently of Y and denotes one or moreamino acid(s)“ Modified-site 31 /label= Y /note= ”Y may be absent orpresent independently of X and denotes one or more amino acid(s)“ 20 XaaTyr Tyr Gln Arg Gly Ser Ser Met Val Leu Phe Ser Ser Pro Pro 1 5 10 15Val Ile Leu Leu Ile Ser Phe Leu Ile Phe Leu Ile Val Gly Xaa 20 25 30 19amino acids amino acid single linear peptide Modified-site 1 /label= X/note= ”X may be absent or present independently of Y and denotes one ormore amino acid(s)“ Modified-site 19 /label= Y /note= ”Y may be absentor present independently of X and denotes one or more amino acid(s)“ 21Xaa Gly Ala Ser Val Ile Leu Phe Ser Ser Pro Pro Val Ile Leu Leu 1 5 1015 Ile Ser Xaa 19 amino acids amino acid single linear peptideModified-site 1 /label= X /note= ”X may be absent or presentindependently of Y and denotes one or more amino acid(s)“ Modified-site19 /label= Y /note= ”Y may be absent or present independently of X anddenotes one or more amino acid(s)“ 22 Xaa Gly Ser Ser Met Val Leu PheSer Ser Pro Pro Val Ile Leu Leu 1 5 10 15 Ile Ser Xaa 33 amino acidsamino acid single linear peptide Modified-site 1 /label= X /note= ”X maybe absent or present independently of Y and denotes one or more aminoacid(s)“ Modified-site 33 /label= Y /note= ”Y may be absent or presentindependently of X and denotes one or more amino acid(s)“ 23 Xaa Pro GlyGly Gly Trp Asn Thr Gly Gly Ser Arg Tyr Pro Gly Gln 1 5 10 15 Gly SerPro Gly Gly Asn Arg Tyr Pro Pro Gln Gly Gly Gly Gly Trp 20 25 30 Xaa 18amino acids amino acid single linear peptide Modified-site 1 /label= X/note= ”X may be absent or present independently of Y and denotes one ormore amino acid(s)“ Modified-site 18 /label= Y /note= ”Y may be absentor present independently of X and denotes one or more amino acid(s)“ 24Xaa Gly Gly Gly Trp Gly Gln Pro His Gly Gly Gly Trp Gly Gln Pro 1 5 1015 His Xaa 30 amino acids amino acid single linear peptide Modified-site1 /label= X /note= ”X may be absent or present independently of Y anddenotes one or more amino acid(s) Modified-site 30 /label= Y /note= “Ymay be absent or present independently of X and denotes one or moreamino acid(s)” 25 Xaa Gly Gly Gly Trp Gly Gln Gly Gly Thr His Gly GlnTrp Asn Lys 1 5 10 15 Pro Ser Lys Pro Lys Thr Asn Met Lys His Val AlaGly Xaa 20 25 30 33 amino acids amino acid single linear peptideModified-site 1 /label= X /note= “X may be absent or presentindependently of Y and denotes one or more amino acid(s)” Modified-site33 /label= Y /note= “Y may be absent or present independently of X anddenotes one or more amino acid(s)” 26 Xaa Pro Gly Gly Gly Trp Asn ThrGly Gly Ser Arg Tyr Pro Gly Gln 1 5 10 15 Gly Ser Pro Gly Gly Asn ArgTyr Pro Pro Gln Gly Gly Gly Gly Trp 20 25 30 Xaa 18 amino acids aminoacid single linear peptide Modified-site 1 /label= X /note= “X may beabsent or present independently of Y and denotes one or more aminoacid(s)” Modified-site 18 /label= Y /note= “Y may be absent or presentindependently of X and denotes one or more amino acid(s)” 27 Xaa Gly GlyGly Trp Gly Gln Pro His Gly Gly Gly Trp Gly Gln Pro 1 5 10 15 His Xaa 30amino acids amino acid single linear peptide Modified-site 1 /label= X/note= “X may be absent or present independently of Y and denotes one ormore amino acid(s)” Modified-site 30 /label= Y /note= “Y may be absentor present independently of X and denotes one or more amino acid(s)” 28Xaa Gly Gly Gly Trp Gly Gln Gly Gly Ser His Ser Gln Trp Asn Lys 1 5 1015 Pro Ser Lys Pro Lys Thr Asn Met Lys His Val Ala Gly Xaa 20 25 30 33amino acids amino acid single linear peptide Modified-site 1 /label= X/note= “X may be absent or present independently of Y and denotes one ormore amino acid(s)” Modified-site 33 /label= Y /note= “Y may be absentor present independently of X and denotes one or more amino acid(s) ” 29Xaa Pro Gly Gly Gly Trp Asn Thr Gly Gly Ser Arg Tyr Pro Gly Gln 1 5 1015 Gly Ser Pro Gly Gly Asn Arg Tyr Pro Pro Gln Gly Gly Gly Gly Trp 20 2530 Xaa 18 amino acids amino acid single linear peptide Modified-site 1/label= X /note= “X is absent or present independently of Y and denotesone or more amino acid(s)” Modified-site 18 /label= Y /note= “Y may beabsent or present independently of X and denotes one or more aminoacid(s)” 30 Xaa Gly Gly Gly Trp Gly Gln Pro His Gly Gly Gly Trp Gly GlnPro 1 5 10 15 His Xaa 31 amino acids amino acid single linear peptideModified-site 1 /label= X /note= “X may be absent or presentindependently of Y and denotes one or more amino acid(s)” Modified-site31 /label= Y /note= “Y may be absent or present independently of X anddenotes one or more amino acid(s)” 31 Xaa Gly Gly Gly Trp Gly Gln GlyGly Gly Thr His Ser Gln Trp Asn 1 5 10 15 Lys Pro Ser Lys Pro Lys ThrAsn Met Lys His Met Ala Gly Xaa 20 25 30 33 amino acids amino acidsingle linear peptide Modified-site 1 /label= X /note= “X may be absentor present independently of Y and denotes one or more amino acid(s)”Modified-site 33 /label= Y /note= “Y may be absent or presentindependently of X and denotes one or more amino acid(s)” 32 Xaa Asn PheVal His Asp Cys Val Asn Ile Thr Val Lys Glu His Thr 1 5 10 15 Val ThrThr Thr Thr Lys Gly Glu Asn Phe Thr Glu Thr Asp Ile Lys 20 25 30 Xaa 22amino acids amino acid single linear peptide Modified-site 1 /label= X/note= “X may be absent or present independently of Y and denotes one ormore amino acid(s)” Modified-site 22 /label= Y /note= “Y may be absentor present independently of X and denotes one or more amino acid(s)” 33Xaa Met Cys Ile Thr Gln Tyr Gln Arg Glu Ser Gln Ala Tyr Tyr Gln 1 5 1015 Arg Gly Ala Ser Val Xaa 20 33 amino acids amino acid single linearpeptide Modified-site 1 /label= X /note= “X may be absent or presentindependently of Y and denotes one or more amino acid(s)” Modified-site33 /label= Y /note= “Y is absent or present independently of X anddenotes one or more amino acid(s)” 34 Xaa Asn Phe Val His Asp Cys ValAsn Ile Thr Val Lys Gln His Thr 1 5 10 15 Val Thr Thr Thr Thr Lys GlyGlu Asn Phe Thr Glu Thr Asp Ile Lys 20 25 30 Xaa 22 amino acids aminoacid single linear peptide Modified-site 1 /label= X /note= “X may beabsent or present independently of Y and denotes one or more aminoacid(s)” Modified-site 22 /label= Y /note= “Y may be absent or presentindependently of X and denotes one or more amino acid(s)” 35 Xaa Met CysIle Thr Gln Tyr Gln Arg Glu Ser Gln Ala Tyr Tyr Gln 1 5 10 15 Arg GlyAla Ser Val Xaa 20 33 amino acids amino acid single linear peptideModified-site 1 /label= X /note= “X may be absent or presentindependently of Y and denotes one or amino acid(s)” Modified-site 33/label= Y /note= “Y may be absent or present independently of X anddenotes one or more amino acid(s)” 36 Xaa Asn Phe Val His Asp Cys ValAsn Ile Thr Ile Lys Gln His Thr 1 5 10 15 Val Thr Thr Thr Thr Lys GlyGlu Asn Phe Thr Glu Thr Asp Val Lys 20 25 30 Xaa 22 amino acids aminoacid single linear peptide Modified-site 1 /label= X /note= “X may beabsent or present independently of Y and denotes one or more aminoacid(s)” Modified-site 22 /label= Y /note= “Y may be absent or presentindependently of X and denotes one or more amino acid(s)” 37 Xaa Met CysIle Thr Gln Tyr Glu Arg Glu Ser Gln Ala Tyr Tyr Gln 1 5 10 15 Arg GlySer Ser Met Xaa 20 5 amino acids amino acid single linear peptide 38 GlyGly Gly Gly Gly 1 5 6 amino acids amino acid single linear peptide 39Gly Pro Gly Pro Gly Pro 1 5 7 amino acids amino acid single linearpeptide 40 Gly Ser Ala Gly Ser Gly Ala 1 5 26 amino acids amino acidsingle linear peptide 41 Ala Met Ser Arg Pro Leu Ile His Phe Gly Ser AspTyr Glu Asp Arg 1 5 10 15 Tyr Tyr Arg Glu Asn Met His Arg Gly Cys 20 2521 amino acids amino acid single linear peptide 42 Ser Ala Met Ser ArgPro Leu Ile His Phe Gly Asn Asp Tyr Glu Asp 1 5 10 15 Arg Tyr Tyr GlyCys 20 21 amino acids amino acid single linear peptide 43 Ser Ala MetSer Arg Pro Leu Ile His Phe Gly Ser Asp Tyr Glu Asp 1 5 10 15 Arg TyrTyr Gly Cys 20 27 amino acids amino acid single linear peptide 44 AsnMet Tyr Arg Tyr Pro Asn Gln Val Tyr Tyr Arg Pro Val Asp Arg 1 5 10 15Tyr Ser Asn Gln Asn Asn Phe Val His Gly Cys 20 25 27 amino acids aminoacid single linear peptide 45 Asn Met His Arg Tyr Pro Asn Gln Val TyrTyr Arg Pro Val Asp Gln 1 5 10 15 Tyr Ser Asn Gln Asn Asn Phe Val HisGly Cys 20 25 26 amino acids amino acid single linear peptide 46 Gly GlnPro His Gly Gly Gly Trp Gly Gln Pro His Gly Gly Gly Trp 1 5 10 15 GlyGln Pro His Gly Gly Gly Trp Gly Cys 20 25 24 amino acids amino acidsingle linear peptide 47 Gly Gln Gly Gly Ser His Ser Gln Trp Asn Lys ProSer Lys Pro Lys 1 5 10 15 Thr Asn Met Lys His Val Gly Cys 20 15 aminoacids amino acid single linear peptide 48 Cys Ile Thr Gln Tyr Gln ArgGlu Ser Gln Ala Tyr Tyr Gln Arg 1 5 10 15 28 amino acids amino acidsingle linear peptide 49 Gly Gly Trp Asn Thr Gly Gly Ser Arg Tyr Pro GlyGln Gly Ser Pro 1 5 10 15 Gly Gly Asn Arg Tyr Pro Pro Gln Gly Gly GlyCys 20 25 23 amino acids amino acid single linear peptide 50 Val Asn IleThr Val Lys Gln His Thr Val Thr Thr Thr Thr Lys Gly 1 5 10 15 Glu AsnPhe Thr Glu Gly Cys 20 29 amino acids amino acid single linear peptide51 Lys His Met Ala Gly Ala Ala Ala Ala Gly Ala Val Val Gly Gly Leu 1 510 15 Gly Gly Tyr Met Leu Gly Ser Ala Met Ser Arg Gly Cys 20 25 33 aminoacids amino acid single linear peptide Modified-site 1 /label= X /note=“X may be absent or present independently of Y and denotes one or aminoacid(s)” Modified-site 2 /label= X /note= “X = either Met, Leu or Phe”Modified-site 5 /label= X /note= “X = Met or Val” Modified-site 11/label= X /note= “X = Ala or absent” Modified-site 31 /label= X /note=“X = either Leu, Ile or Met” Modified-site 32 /label= X /note= “X = Leu,Ile or Met” Modified-site 33 /label= Y /note= “Y may be absent orpresent independently of X and denotes one or more amino acid(s)” 52 XaaXaa Lys His Xaa Ala Gly Ala Ala Ala Xaa Gly Ala Val Val Gly 1 5 10 15Gly Leu Gly Gly Tyr Met Leu Gly Ser Ala Met Ser Arg Pro Xaa Xaa 20 25 30Xaa 31 amino acids amino acid single linear peptide Modified-site 1/label= X /note= “X may be absent or present independently of Y anddenotes one or more amino acid(s)” Modified-site 8 /label= X /note= “X =either Leu, Ile, or Met” Modified-site 9 /label= X /note= “X = eitherLeu, Ile or Met” Modified-site 13 /label= X /note= “X = Asn or Ser”Modified-site 15 /label= X /note= “X = Tyr or Trp” Modified-site 25/label= X /note= “X = either His, Tyr or Asn” Modified-site 31 /label= Y/note= “Y may be absent or present independently of X and denotes one ormore amino acid(s)” 53 Xaa Ser Ala Met Ser Arg Pro Xaa Xaa His Phe GlyXaa Asp Xaa Glu 1 5 10 15 Asp Arg Tyr Tyr Arg Glu Asn Met Xaa Arg TyrPro Asn Gln Xaa 20 25 30 31 amino acids amino acid single linear peptideModified-site 1 /label= X /note= “X may be absent or presentindependently of Y and denotes one or more amino acid(s)” Modified-site4 /label= X /note= “X = either His, Tyr or Asn” Modified-site 15 /label=X /note= “X = Val or Met” Modified-site 17 /label= X /note= “X = eitherGln, Glu or Arg” Modified-site 19 /label= X /note= “X = Ser or Asn”Modified-site 31 /label= Y /note= “Y may be absent or presentindependently of X and denotes one or more amino acid(s)” 54 Xaa Asn MetXaa Arg Tyr Pro Asn Gln Val Tyr Tyr Arg Pro Xaa Asp 1 5 10 15 Xaa TyrXaa Asn Gln Asn Asn Phe Val His Asp Cys Val Asn Xaa 20 25 30 33 aminoacids amino acid single linear peptide Modified-site 1 /label= X /note=“X may be absent or present independently of Y and denotes one or moreamino acid(s)” Modified-site 4 /label= X /note= “X = Asp or Gln”Modified-site 5 /label= X /note= “X = Gly or absent” Modified-site 7/label= X /note= “X = Gly or Arg” Modified-site 8 /label= X /note= “X =Ala or Ser” Modified-site 10 /label= X /note= “X = Ser or absent”Modified-site 11 /label= X /note= “X = Ala, Thr, Met or Val”Modified-site 12 /label= X /note= “X = Val or Ile” Modified-site 30/label= X /note= “X= Ile or Met” Modified-site 33 /label= Y /note= “Ymay be absent or present independently of X and denotes one or moreamino acid(s)” 55 Xaa Tyr Tyr Xaa Xaa Arg Xaa Xaa Ser Xaa Xaa Xaa LeuPhe Ser Ser 1 5 10 15 Pro Pro Val Ile Leu Leu Ile Ser Phe Leu Ile PheLeu Xaa Val Gly 20 25 30 Xaa 33 amino acids amino acid single linearpeptide Modified-site 1 /label= X /note= “X may be absent or presentindependently of Y and denotes one or more amino acid(s)” Modified-site5 /label= X /note= “X = Gly or absent” Modified-site 30 /label= X /note=“X = Gly or absent” Modified-site 31 /label= X /note= “X = Gly or Thr”Modified-site 33 /label= Y /note= “Y may be absent or presentindependently of X and denotes one or more amino acid(s)” 56 Xaa Pro GlyGly Xaa Trp Asn Thr Gly Gly Ser Arg Tyr Pro Gly Gln 1 5 10 15 Gly SerPro Gly Gly Asn Arg Tyr Pro Pro Gln Gly Gly Xaa Xaa Trp 20 25 30 Xaa 20amino acids amino acid single linear peptide Modified-site 1 /label= X/note= “X may be absent or present independently of Y and denotes one ormore amino acid(s)” Modified-site 4 /label= X /note= “X = Gly or absent”Modified-site 5 /label= X /note= “X = Gly or Thr” Modified-site 14/label= X /note= “X = Gly or absent” Modified-site 20 /label= Y /note=“Y may be absent or present independently of X and denotes on or moreamino acid(s)” 57 Xaa Gly Gly Xaa Xaa Trp Gly Gln Pro His Gly Gly GlyXaa Trp Gly 1 5 10 15 Gln Pro His Xaa 20 31 amino acids amino acidsingle linear peptide Modified-site 1 /label= X /note= “X may be absentor present independently of Y and denotes one or more amino acid(s)”Modified-site 10 /label= X /note= “X = Gly or absent” Modified-site 11/label= X /note= “X = Thr or Ser” Modified-site 13 /label= X /note= “X =either Gly, Ser or Asn” Modified-site 19 /label= X /note= “X = Asn orSer” Modified-site 24 /label= X /note= “X = Asn or Ser” Modified-site 25/label= X /note= “X = either Met, Leu or Phe” Modified-site 28 /label= X/note= “X = Val or Met” Modified-site 31 /label= Y /note= “Y may beabsent or present independently of X and denotes one or more aminoacid(s)” 58 Xaa Gly Gly Gly Trp Gly Gln Gly Gly Xaa Xaa His Xaa Gln TrpAsn 1 5 10 15 Lys Pro Xaa Lys Pro Lys Thr Xaa Xaa Lys His Xaa Ala GlyXaa 20 25 30 18 amino acids amino acid single linear peptideModified-site 1 /label= X /note= “X may be absent or presentindependently of Y and denotes one or more amino acid(s)” Modified-site2 /label= X /note= “X = Ala or Thr” Modified-site 3 /label= X /note= “X= either Ser, Pro or Thr” Modified-site 5 /label= X /note= “X = Trp orArg” Modified-site 9 /label= X /note= “X = either, Ala, Ser, Pro, andThr” Modified-site 10 /label= X /note= “X = Ala or Thr” Modified-site 11/label= X /note= “X = either Ser, Pro or Thr” Modified-site 13 /label= X/note= “X = Trp or Arg” Modified-site 17 /label= X /note= “X = eitherAla, Ser, Pro, or Thr” Modified-site 18 /label= Y /note= “Y may beabsent or present independently of X and denotes one or more aminoacid(s)” 59 Xaa Xaa Xaa Trp Xaa Trp Leu Gly Xaa Xaa Xaa Trp Xaa Trp LeuGly 1 5 10 15 Xaa Xaa 18 amino acids amino acid single linear peptideModified-site 1 /label= X /note= “X may be absent or presentindependently of Y and denotes one or more amino acid(s)” Modified-site2 /label= X /note= “X = Ser or Asn” Modified-site 3 /label= X /note= “X= either Pro, Leu or His” Modified-site 5 /label= X /note= “X = Val orGlu” Modified-site 9 /label= X /note= “X = either Val, Ala, Asp or Gly”Modified-site 10 /label= X /note= “X = Ser or Asn” Modified-site 11/label= X /note= “X = either Pro, Leu or His” Modified-site 13 /label= X/note= “X = Val or Glu” Modified-site 17 /label= X /note= “X = eitherVal, Ala, Asp or Gly” Modified-site 18 /label= Y /note= “Y may be absentor present independently of X and denotes one or more amino acid(s)” 60Xaa Xaa Xaa Met Xaa Val Ala Gly Xaa Xaa Xaa Met Xaa Val Ala Gly 1 5 1015 Xaa Xaa 33 amino acids amino acid single linear peptide Modified-site1 /label= X /note= “X may be absent or present independently of Y anddenotes one or more amino acid(s)” Modified-site 12 /label= X /note= “X= Ile or Val” Modified-site 14 /label= X /note= “X = Gln or Glu”Modified-site 18 /label= X /note= “X = Val or Thr” Modified-site 31/label= X /note= “X = Val or Ile” Modified-site 33 /label= Y /note= “Ymay be absent or present independently of X and denotes one or moreamino acid(s)” 61 Xaa Asn Phe Val His Asp Cys Val Asn Ile Thr Xaa LysXaa His Thr 1 5 10 15 Val Xaa Thr Thr Thr Lys Gly Glu Asn Phe Thr GluThr Asp Xaa Lys 20 25 30 Xaa 24 amino acids amino acid single linearpeptide Modified-site 1 /label= X /note= “X may be absent or presentindependently of Y and denotes one or more amino acid(s)” Modified-site4 /label= X /note= “X = either Ile, Thr or Val” Modified-site 8 /label=X /note= “X = Gln or Glu” Modified-site 9 /label= X /note= “X = Arg orLys” Modified-site 16 /label= X /note= “X = Asp or Gln” Modified-site 17/label= X /note= “X = Gly or absent” Modified-site 19 /label= X /note=“X = Gly or Arg” Modified-site 20 /label= X /note= “X = Ala or Ser”Modified-site 22 /label= X /note= “X = Ser or absent” Modified-site 23/label= X /note= “X = either Ala, Thr, Met or Val” Modified-site 24/label= Y /note= “Y may be absent or present independently of X anddenotes one or more amino acid(s)” 62 Xaa Met Cys Xaa Thr Gln Tyr XaaXaa Glu Ser Gln Ala Tyr Tyr Xaa 1 5 10 15 Xaa Arg Xaa Xaa Ser Xaa XaaXaa 20 18 amino acids amino acid single linear peptide Modified-site 1/label= X /note= “X may be absent or present independently of Y anddenotes one or more amino acid(s)” Modified-site 3 /label= X /note= “X =Met or Val” Modified-site 9 /label= X /note= “X = Ala or absent”Modified-site 18 /label= Y /note= “Y may be absent or presentindependently of X and denotes one or more amino acid(s)” 63 Xaa His XaaAla Gly Ala Ala Ala Xaa Gly Ala Val Val Gly Gly Leu 1 5 10 15 Gly Xaa 19amino acids amino acid single linear peptide Modified-site 1 /label= X/note= “X may be absent or present independently of Y and denotes one ormore amino acid(s)” Modified-site 17 /label= X /note= “X = either Leu,Ile, or Met” Modified-site 18 /label= X /note= “X = either Leu, Ile, orMet” Modified-site 19 /label= Y /note= “Y may be absent or presentindependently of X and denotes one or more amino acid(s)” 64 Xaa Gly GlyLeu Gly Gly Tyr Met Leu Gly Ser Ala Met Ser Arg Pro 1 5 10 15 Xaa XaaXaa 25 amino acids amino acid single linear peptide Modified-site 1/label= X /note= “X may be absent or present independently of Y anddenotes one or more amino acid(s)” Modified-site 8 /label= X /note= “X =either Leu, Ile or Met” Modified-site 9 /label= X /note= “X = eitherLeu, Ile or Met” Modified-site 13 /label= X /note= “X = Asn or Ser”Modified-site 15 /label= X /note= “X = Tyr or Trp” Modified-site 25/label= /note= “Y may be absent or present independently of X anddenotes one or more amnio acid(s)” 65 Xaa Ser Ala Met Ser Arg Pro XaaXaa His Phe Gly Xaa Asp Xaa Glu 1 5 10 15 Asp Arg Tyr Tyr Arg Glu AsnMet Xaa 20 25 28 amino acids amino acid single linear peptideModified-site 1 /label= X /note= “X may be absent or presentindependently of Y and denotes one or more amino acid(s)” Modified-site12 /label= X /note= “X = Val or Met” Modified-site 14 /label= X /note=“X = either Gln, Glu or Arg” Modified-site 16 /label= X /note= “X = Seror Asn” Modified-site 28 /label= Y /note= “Y may be absent or presentindependently of X and denotes one or more amino acid(s)” 66 Xaa Arg TyrPro Asn Gln Val Tyr Tyr Arg Pro Xaa Asp Xaa Tyr Xaa 1 5 10 15 Asn GlnAsn Asn Phe Val His Asp Cys Val Asn Xaa 20 25 20 amino acids amino acidsingle linear peptide Modified-site 1 /label= X /note= “X may be absentor present independently of Y and denotes one or more amino acid(s)”Modified-site 2 /label= X /note= “X = Gly or Arg” Modified-site 3/label= X /note= “X = Ala or Ser” Modified-site 5 /label= X /note= “X =Ser or absent” Modified-site 6 /label= X /note= “X = either Ala, Thr,Met or Val” Modified-site 7 /label= X /note= “X = Val or Ile”Modified-site 20 /label= Y /note= “Y may be absent or presentindependently of X and denotes one or more amino acid(s)” 67 Xaa Xaa XaaSer Xaa Xaa Xaa Leu Phe Ser Ser Pro Pro Val Ile Leu 1 5 10 15 Leu IleSer Xaa 20

What is claimed is:
 1. A synthetic polypeptide having at least oneantigenic site of a prion protein, wherein the polypeptide is selectedfrom the group consisting of polypeptides of Formulae I, II, III, IV,VI, VII, VIIIa, VIIIb and IX, or has one of the following sequences:(Formula I (SEQ ID NO:52))X-(R₁-Lys-His-R₂)-Ala-Gly-Ala-Ala-Ala-R₃-Gly-Ala-Val-Val-Gly-Gly-Leu-Gly-Gly-Tyr-Met-Leu-Gly-Ser-Ala-Met-Ser-(Arg-Pro-R₄-R₅)-Y

wherein R₁ is an amino acid residue selected from Met, Leu and Phe; R₂is either Met or Val; R₃ is Ala or is absent; R₄ and R₅ areindependently an amino acid residue selected from Leu, Ile and Met;(Formula II (SEQ ID NO:53))X-(Ser-Ala-Met-Ser)-Arg-Pro-R₄-R₅-His-Phe-Gly-R₆-Asp-R₇-Glu-Asp-Arg-Tyr-Tyr-Arg-Glu-Asn-Met-R₈-Arg- (Tyr-Pro-Asn-Gln)-Y

wherein R₄ and R₅ are the same as in Formula I; R₆ is either Asn or Ser;R₇ is either Tyr or Trp; R₈ is an amino acid residue selected from His,Tyr and Asn; (Formula III (SEQ ID NO:54))X-(Asn-Met-R₈-Arg)-Tyr-Pro-Asn-Gln-val-Tyr-Tyr-Arg-Pro-R₉-Asp-R₁₀-Tyr-R₁₁-Asn-Gln-Asn-Asn-Phe-Val-His-(Asp-Cys-Val-Asn)-Y

wherein R₈ is an amino acid residue selected from His, Tyr and Asn; R₉is Val or Met; R₁₀ is an amino acid residue selected from Gln, Glu andArg; R₁₁ is Ser or Asn; (Formula IV (SEQ ID NO:55))X-(Tyr-Tyr-R₁₂-R₁₃-Arg)-R₁₄-R₁₅-Ser-R₁₆-R₁₇-R₁₈-Leu-Phe-Ser-Ser-Pro-Pro-Val-Ile-Leu-Leu-Ile-Ser-Phe-Leu-Ile-Phe-Leu-R₁₉-Val-Gly-Y

wherein R₁₂ is Asp or Gln; R₁₃ is Gly or absent; R₁₄ is Gly or Arg; R₁₅is Ala or Ser; R₁₆ is Ser or absent; R₁₇ is an amino acid residueselected from Ala, Thr, Met and Val; R₁₈ is Val or Ile; R₁₉ is Ile orMet; (Formula VI (SEQ ID NO:59))X-(R₃₁-R₃₂-Trp-R₃₃)-Trp-Leu-Gly-R₃₄-R₃₅-R₃₆-Trp- R₃₇(Trp-Leu-Gly-R₃₈)-Y

wherein R₃₁ and R₃₅ are each independently either Ala or Thr; R₃₂ andR₃₆ are each independently an amino acid residue selected from Ser, Proand Thr; R₃₃ and R₃₇ are each independently either Trp or Arg; R₃₄ andR₃₈ are each independently an amino acid residue selected from Ala, Ser,Pro and Thr; (Formula VII (SEQ ID NO:60))X-(R₃₉-R₄₀-Met-R₄₁)-Val-Ala-Gly-R₄₂-R₄₃-R₄₄-Met-R₄₅- (Val-Ala-Gly-R₄₆)-Y

wherein R₃₉ and R₄₃ are each independently either Ser or Asn; R₄₀ andR₄₄ are each independently an amino acid residue selected from Pro, Leuand His, R₄₁ and R₄₅ are each independently Val or Glu; R₄₂ and R₄₆ areeach independently selected from Val, Ala, Asp and Gly; (Formula VIIIa(SEQ ID NO:61)) X-(Asn-Phe-Val-His)-Asp-Cys-Val-Asn-Ile-Thr-R₄₇-Lys-R₄₈-His-Thr-Val-R₄₉-Thr-Thr-Thr-Lys-Gly-Glu-Asn-Phe-Thr-Glu-(Thr-Asp-R₅₀-Lys)-Y

wherein R₄₇ is either Ile or Val; R₄₈ is either Gln or Glu; R₄₉ iseither Val or Thr; R₅₀ is either Val or Ile; (Formula VIIIb (SEQ IDNO:62)) X-(Met-Cys-R₅₁-Thr)-Gln-Tyr-R₅₂-R₅₃-Glu-Ser-Gln-Ala-Tyr-Tyr-R₅₄-R₅₅-Arg-(R₅₆-R₅₇-Ser-R₅₈-R₅₉)-Y

wherein R₅₁ is an amino acid residue selected from Ile, Thr and Val; R₅₂is either Gln or Glu; R₅₃ is either Arg or Lys; R₅₄ is either Asp orGln; R₅₅ is Gly or is absent; R₅₆ is either Gly or Arg; R₅₇ is eitherAla or Ser; R₅₈ is Ser or absent; R₅₉ is an amino acid residue selectedfrom Ala, Thr, Met and Val; wherein one or more residues within theparentheses are present or absent with the proviso that if they arepresent they are attached to the rest of the peptide in sequence; and Xand Y are each independently absent or one or more additional amino acidresidues, with the proviso that when present neither X nor Y provide orform part of an antigenic property of the prion protein which, in thecorresponding portion of sequence of a natural prion protein, iscontiguous with the sequence to which X and Y are attached; or [L _(a)−F] _(m) −[L _(b) −G] _(n) −L _(c)  (Formula IX) wherein F and G areeach independently a polypeptide according to any one of Formulae I toIV and VI to VIIIb, L is a linking sequence, a, b and c are eachindependently 0 or 1, and m and n are each positive numbers, or F and Gare each independently a subfragment of such sequence.
 2. A syntheticpolypeptide as claimed in claim 1 comprising a sequence according togeneral Formula (I): (Formula I (SEQ ID NO:52))X-(R₁-Lys-His-R₂)-Ala-Gly-Ala-Ala-Ala-R₃-Gly-Ala-Val-Val-Gly-Gly-Leu-Gly-Gly-Tyr-Met-Leu-Gly-Ser-Ala-Met-Ser-(Arg-Pro-R₄-R₅)-Y

wherein R₁ is an amino acid residue selected from Met, Leu and Phe; R₂is either Met or Val; R₃ is Ala or is absent; R₄ and R₅ areindependently an amino acid residue selected from Leu, Ile and Met; oneor more residues within the parentheses are present or absent with theproviso that if they are present they are attached to the rest of thepeptide in sequence; and X and Y are each independently absent or one ormore additional amino acid residues, with the proviso that when presentneither X nor Y provide or form part of an antigenic property of theprion protein which, in the corresponding portion of sequence of anatural prion protein, is contiguous with the sequence to which X and Yare attached.
 3. A synthetic polypeptide as claimed in claim 2 havingthe sequence Seq. I.D. No:1X-(Met-Lys-His-Val)-Ala-Gly-Ala-Ala-Ala-Ala-Gly-Ala-Val-Val-Gly-Gly-Leu-Gly-Gly-Tyr-Met-Leu-Gly-Ser-Ala-Met-Ser-(Arg-Pro-Leu-Ile)-Y; or Seq. I.D. No:2X-(Met-Lys-His-Met)-Ala-Gly-Ala-Ala-Ala-Ala-Gly-Ala-Val-Val-Gly-Gly-Leu-Gly-Gly-Tyr-Met-Leu-Gly-Ser-Ala-Met-Ser-(Arg-Pro-Ile-Ile)-Y.


4. A synthetic polypeptide as claimed in claim 2 having the sequenceSeq. I.D. No:51 Lys-His-Net-Ala-Gly-Ala-Ala-Ala-Ala-Gly-Ala-Val-Val-Gly-Gly-Leu-Gly-Gly-Tyr-Met-Leu-Gly-Ser-Ala- Met-Ser-Arg-Gly-Cys.


5. A significant sub-fragment of a sequence claimed in claim 2 havingthe sequence Seq. I.D. No: 63X-(His-R₂-Ala-Gly)-Ala-Ala-Ala-R₃-Gly-Ala-Val-Val- (Gly-Gly-Leu-Gly)-Y;or Seq. I.D. No: 64 X-(Gly-Gly-Leu-Gly)-Gly-Tyr-Met-Leu-Gly-Ser-Ala-Met-Ser-(Arg-Pro-R₄-R₅)-Y

wherein R₂, R₃, R₄, R₅, X and Y are as defined in Formula I and one ormore residues in the parentheses are absent or present as in Formula I.6. A sub-fragment as claimed in claim 5 having the sequence Seq. I.D.No: 3 i) X-(His-Val-Ala-Gly)-Ala-Ala-Ala-Ala-Gly-Ala-Val-Val-Gly-(Gly-Leu-Gly-Gly)-Y; Seq. I.D. No: 4 ii)X-(Gly-Gly-Leu-Gly)-Gly-Tyr-Met-Leu-Gly-Ser-Ala-Met-Ser-(Arg-Pro-Leu-Ile)-Y Seq. I.D. No: 5 i)X-(His-Met-Ala-Gly)-Ala-Ala-Ala-Ala-Gly-Ala-Val-Val-Gly-(Gly-Leu-Gly-Gly)-Y; or Seq. I.D. No: 6 ii)X-(Gly-Gly-Leu-Gly)-Gly-Tyr-Met-Leu-Gly-Ser-Ala-Met-Ser-(Arg-Pro-Ile-Ile)-Y.


7. A synthetic polypeptide as claimed in claim 1 having a sequenceaccording to general Formula II: (SEQ ID NO:53)X-(Ser-Ala-Met-Ser)-Arg-Pro-R₄-R₅-His-Phe-Gly-R₆-Asp-R₇-Glu-Asp-Arg-Tyr-Tyr-Arg-Glu-Asn-Met-R₈-Arg- (Tyr-Pro-Asn-Gln)-Y(Formula II)

wherein R₄ and R₅ are the same as in Formula I; R₆ is either Asn or Ser;R₇ is either Tyr or Trp; R₈ is an amino acid residue selected from His,Tyr and Asn; one or more residues within the parentheses are present orabsent with the proviso that if they are present they are attached tothe rest of the peptide in sequence; and X and Y are each independentlyabsent or one or more additional amino acid residues, with the provisothat when present neither X nor Y provide or form part of an antigenicproperty of the prion protein which, in the corresponding portion ofsequence of a natural prion protein, is contiguous with the sequence towhich X and Y are attached.
 8. A synthetic polypeptide as claimed inclaim 7 having the sequence Seq. I.D. No: 7X-(Ser-Ala-Met-Ser)-Arg-Pro-Leu-Ile-His-Phe-Gly-Ser-Asp-Tyr-Glu-Asp-Arg-Tyr-Tyr-Arg-Glu-Asn-Met-His-Arg-(Tyr-Pro-Asn-Gln)-Y; Seq. I.D. No: 8X-(Ser-Ala-Met-Ser)-Arg-Pro-Leu-Ile-His-Phe-Gly-Asn-Asp-Tyr-Glu-Asp-Arg-Tyr-Tyr-Arg-Glu-Asn-Met-Tyr-Arg-(Tyr-Pro-Asn-Gln)-Y; or Seq. I.D. No: 9X-(Ser-Ala-Met-Ser)-Arg-Pro-Ile-Ile-His-Phe-Gly-Ser-Asp-Tyr-Glu-Asp-Arg-Tyr-Tyr-Arg-Glu-Asn-Met-His-Arg-(Tyr-Pro-Asn-Gln)-Y.


9. A significant sub-fragment of a sequence as claimed in claim 7 havingthe sequence: X-(Ser-Ala-Met-Ser)-Arg-Pro-R₄-R₅-His-Phe-Gly-R₆-Asp-R₇-Glu-Asp-Arg-Tyr-Tyr-(Arg-Glu-Asn-Met)-Y

wherein R₄ to R₇, X and Y are as defined in Formula II and one or moreresidues in parentheses are present or absent.
 10. A syntheticpolypeptide as claimed in claim 9 having the sequence Seq. I.D. No: 42Ser-Ala-Met-Ser-Arg-Pro-Leu-Ile-His-Phe-Gly-Asn-Asp-Tyr-Glu-Asp-Arg-Tyr-Tyr-Gly-Cys; or Seq. I.D. No: 43Ser-Ala-Met-Ser-Arg-Pro-Leu-Ile-His-Phe-Gly-Ser-Asp-Tyr-Glu-Asp-Arg-Tyr-Tyr-Gly-Cys.


11. A sub-fragment as claimed in claim 9 having the sequence Seq. I.D.No: 10 X-(Ser-Ala-Met-Ser)-Arg-Pro-Leu-Ile-His-Phe-Gly-Ser-Asp-Tyr-Glu-Asp-Arg-Tyr-Tyr-(Arg-Glu-Asn- Met)-Y; Seq. I.D. No: 11X-(Ser-Ala-Met-Ser)-Arg-Pro-Leu-Ile-His-Phe-Gly-Asn-Asp-Tyr-Glu-Asp-Arg-Tyr-Tyr-(Arg-Glu-Asn-Met)- Y; or Seq. I.D. No:12 X-(Ser-Ala-Met-Ser)-Arg-Pro-Ile-Ile-His-Phe-Gly-Ser-Asp-Tyr-Glu-Asp-Arg-Tyr-Tyr-(Arg-Glu-Asn-Met)- Y.


12. A synthetic polypeptide as claimed in claim 1 having a sequenceaccording to general Formula III: (SEQ ID NO:54)X-(Asn-Met-R₈-Arg)-Tyr-Pro-Asn-Gln-Val-Tyr-Tyr-Arg-Pro-R₉-Asp-R₁₀-Tyr-R₁₁-Asn-Gln-Asn-Asn-Phe-Val-His-(Asp-Cys-Val-Asn)-Y (Formula III)

wherein R₈ is an amino acid residue selected from His, Tyr and Asn; R₉is Val or Met; R₁₀ is an amino acid residue selected from Gln, Glu andArg; R₁₁ is Ser or Asn; one or more residues within the parentheses arepresent or absent with the proviso that if they are present they areattached to the rest of the peptide in sequence; and X and Y are eachindependently absent or one or more additional amino acid residues, withthe proviso that when present neither X nor Y provide or form part of anantigenic property of the prion protein which, in the correspondingportion of sequence of a natural prion protein, is contiguous with thesequence to which X and Y are attached.
 13. A synthetic polypeptide asclaimed in claim 12 having the sequence Seq. I.D. No: 13X-(Asn-Met-His-Arg)-Tyr-Pro-Asn-Gln-Val-Tyr-Tyr-Arg-Pro-Val-Asp-Gln-Tyr-Ser-Asn-Gln-Asn-Asn-Phe-Val-His-(Asp-Cys-Val-Asn)-Y; Seq. I.D. No: 14X-(Asn-Met-Tyr-Arg)-Tyr-Pro-Asn-Gln-Val-Tyr-Tyr-Arg-Pro-Val-Asp-Arg-Tyr-Ser-Asn-Gln-Asn-Asn-Phe-Val-His-(Asp-Cys-Val-Asn)-Y; or Seq. I.D. No: 15X-(Asn-Met-His-Arg)-Tyr-Pro-Asn-Gln-Val-Tyr-Tyr-Arg-Pro-Met-Asp-Glu-Tyr-Ser-Asn-Gln-Asn-Asn-Phe-Val-His-(Asp-Cys-Val-Asn)-Y.


14. A synthetic polypeptide as claimed in claim 13 having the sequenceSeq. I.D. No: 44 Asn-Met-Tyr-Arg-Tyr-Pro-Asn-Gln-Val-Tyr-Tyr-Arg-Pro-Val-Asp-Arg-Tyr-Ser-Asn-Gln-Asn-Asn-Phe-Val- His-Gly-Cys; or Seq.I.D. No: 45 Asn-Met-His-Arg-Tyr-Pro-Asn-Gln-Val-Tyr-Tyr-Arg-Pro-Val-Asp-Gln-Tyr-Ser-Asn-Gln-Asn-Asn-Phe-Val- His-Gly-Cys.


15. A significant sub-fragment of a sequence as claimed in claim 12having the sequence: Seq. I.D. No:66X-(Arg-Tyr-Pro-Asn)-Gln-Val-Tyr-Tyr-Arg-Pro-R₉-Asp-R₁₀-Tyr-R₁₁-Asn-Gln-Asn-Asn-Phe-Val-His- (Asp-Cys-Val-Asn)-Y

wherein R₉, R₁₀, R₁₁, X and Y are as defined in Formula (III).
 16. Asub-fragment as claimed in claim 15 having the sequence Seq. I.D. No: 16X-(Arg-Tyr-Pro-Asn)-Gln-Val-Tyr-Tyr-Arg-Pro-Val-Asp-Gln-Tyr-Ser-Asn-Gln-Asn-Asn-Phe-Val-His- (Asp-Cys-Val-Asn)-Y; Seq.I.D. No: 17 X-(Arg-Tyr-Pro-Asn)-Gln-Val-Tyr-Tyr-Arg-Pro-Val-Asp-Arg-Tyr-Ser-Asn-Gln-Asn-Asn-Phe-Val-His- (Asp-Cys-Val-Asn)-Y; orSeq. I.D. No: 18 X-(Arg-Tyr-Pro-Asn)-Gln-Val-Tyr-Tyr-Arg-Pro-Met-Asp-Glu-Tyr-Ser-Asn-Gln-Asn-Asn-Phe-Val-His- (Asp-Cys-Val-Asn)-Y.


17. A synthetic polypeptide as claimed in claim 1 having a sequenceaccording to general Formula IV: (SEQ ID NO:55)X-(Tyr-Tyr-R₁₂-R₁₃-Arg)-R₁₄-R₁₅-Ser-R₁₆-R₁₇-R₁₈-Leu-Phe-Ser-Ser-Pro-Pro-Val-Ile-Leu-Leu-Ile-Ser-Phe-Leu-Ile-Phe-Leu-R₁₉-Val-Gly-Y (Formula IV)

wherein R₁₂ is Asp or Gln; R₁₃ is Gly or absent; R₁₄ is Gly or Arg; R₁₅is Ala or Ser; R₁₆ is Ser or absent; R₁₇ is an amino acid residueselected from Ala, Thr, Met and Val; R₁₈ is Val or Ile; R₁₉ is Ile orMet; one or more residues within the parentheses are present or absentwith the proviso that if they are present they are attached to the restof the peptide in sequence; and X and Y are each independently absent orone or more additional amino acid residues, with the proviso that whenpresent neither X nor Y provide or form part of an antigenic property ofthe prion protein which, in the corresponding portion of sequence of anatural prion protein, is contiguous with the sequence to which X and Yare attached.
 18. A synthetic polypeptide as claimed in claim 17 havingthe sequence Seq. I.D. No: 19X-(Tyr-Tyr-Gln-Arg)-Gly-Ala-Ser-Val-Ile-Leu-Phe-Ser-Ser-Pro-Pro-Val-Ile-Leu-Leu-Ile-Ser-Phe-Leu-Ile-Phe-Leu-Ile-Val-Gly-Y; or Seq. I.D. No: 20X-(Tyr-Tyr-Gln-Arg)-Gly-Ser-Ser-Met-Val-Leu-Phe-Ser-Ser-Pro-Pro-Val-Ile-Leu-Leu-Ile-Ser-Phe-Leu-Ile-Phe-Leu-Ile-Val-Gly-Y.


19. A significant sub-fragment of a sequence as claimed in claim 17having the sequence: Seq. I.D. No: 67X-(R₁₄-R₁₅-Ser-R₁₆-R₁₇)-R₁₈-Leu-Phe-Ser-Ser-Pro-Pro-Val-Ile-(Leu-Leu-Ile-Ser)-Y

wherein R₁₄ to R₁₈, X and Y are as defined in Formula IV and one or moreresidues within the parentheses are present or absent as in Formula IV.20. A sub-fragment as claimed in claim 19 having the sequence Seq. I.D.No: 21 X-(Gly-Ala-Ser-Val)-Ile-Leu-Phe-Ser-Ser-Pro-Pro-Val-Ile-(Leu-Leu-Ile-Ser)-Y; or Seq. I.D. No: 22X-(Gly-Ser-Ser-Met)-Val-Leu-Phe-Ser-Ser-Pro-Pro-Val-Ile-(Leu-Leu-Ile-Ser)-Y.


21. A synthetic polypeptide as claimed in claim 1 having a sequenceaccording to Formula VI: (SEQ ID NO:59)X-(R₃₁-R₃₂-Trp-R₃₃)-Trp-Leu-Gly-R₃₄-R₃₅-R₃₆-Trp- R₃₇(Trp-Leu-Gly-R₃₈)-Y(Formula VI)

wherein R₃₁ and R₃₅ are each independently either Ala or Thr; R₃₂ andR₃₆ are each independently an amino acid residue selected from Ser, Proand Thr; R₃₃ and R₃₇ are each independently either Trp or Arg; R₃₄ andR₃₈ are each independently an amino acid residue selected from Ala, Ser,Pro and Thr; one or more residues within the parentheses are present orabsent with the proviso that if they are present they are attached tothe rest of the peptide in sequence; and X and Y are each independentlyabsent or one or more additional amino acid residues, with the provisothat when present neither X nor Y provide or form part of an antigenicproperty of the prion protein which, in the corresponding portion ofsequence of a natural prion protein, is contiguous with the sequence towhich X and Y are attached.
 22. A synthetic polypeptide as claimed inclaim 1 having a sequence according to Formula VII: (SEQ ID NO:60)X-(R₃₉-R₄₀-Met-R₄₁)-Val-Ala-Gly-R₄₂-R₄₃-R₄₄-Met- R₄₅-(Val-Ala-Gly-R₄₆)-Y(Formula VII)

wherein R₃₉ and R₄₃ are each independently either Ser or Asn; R₄₀ andR₄₄ are each independently an amino acid residue selected from Pro, Leuand His; R₄₁ and R₄₅ are each independently Val or Glu; R₄₂ and R₄₆ areeach independently selected from Val, Ala, Asp and Gly; one or moreresidues within the parentheses are present or absent with the provisothat if they are present they are attached to the rest of the peptide insequence; and X and Y are each independently absent or one or moreadditional amino acid residues, with the proviso that when presentneither X nor Y provide or form part of an antigenic property of theprion protein which, in the corresponding portion of sequence of anatural prion protein, is contiguous with the sequence to which X and Yare attached.
 23. A synthetic polypeptide as claimed in claim 1 having asequence according to Formulae VIIIa or VIIIb: (SEQ ID NO:61)X-(Asn-Phe-Val-His)-Asp-Cys-Val-Asn-Ile-Thr-R₄₇-Lys-R₄₈-His-Thr-Val-R₄₉-Thr-Thr-Thr-Lys-Gly-Glu-Asn-Phe-Thr-Glu-(Thr-Asp-R₅₀-Lys)-Y (Formula VIIIa)

wherein R₄₇ is either Ile or Val; R₄₈ is either Gln or Glu; R₄₉ iseither Val or Thr; R₅₀ is either Val or Ile; (SEQ ID NO:62)X-(Met-Cys-R₅₁-Thr)-Gln-Tyr-R₅₂-R₅₃-Glu-Ser-Gln-Ala-Tyr-Tyr-R₅₄-R₅₅-Arg-(R₅₆-R₅₇-Ser-R₅₈-R₅₉)-Y (Formula VIIIb)

wherein R₅₁ is an amino acid residue selected from Ile, Thr and Val; R₅₂is either Gln or Glu; R₅₃ is either Arg or Lys; R₅₄ is either Asp orGln; R₅₅ is Gly or is absent; R₅₆ is either Gly or Arg; R₅₇ is eitherAla or Ser; R₅₈ is Ser or absent; R₅₉ is an amino acid residue selectedfrom Ala, Thr, Met and Val; wherein one or more residues within theparentheses of Formulae VIIIa or VIIIb are present or absent with theproviso that if they are present they are attached to the rest of thepeptide in sequence; and X and Y are each independently absent or one ormore additional amino acid residues, with the proviso that when presentneither X nor Y provide or form part of an antigenic property of theprion protein which, in the corresponding portion of sequence of anatural prion protein, is contiguous with the sequence to which X and Yare attached.
 24. A synthetic polypeptide as claimed in claim 23 havingthe sequence Seq I.D. No: 32X-(Asn-Phe-Val-His)-Asp-Cys-Val-Asn-Ile-Thr-Val-Lys-Glu-His-Thr-Val-Thr-Thr-Thr-Thr-Lys-Gly-Glu-Asn-Phe-Thr-Glu-(Thr-Asp-Ile-Lys)-Y; Seq. I.D. No: 33X-(Met-Cys-Ile-Thr)-Gln-Tyr-Gln-Arg-Glu-Ser-Gln-Ala-Tyr-Tyr-Gln-Arg-(Gly-Ala-Ser-Val)-Y; Seq. I.D. No: 34X-(Asn-Phe-Val-His)-Asp-Cys-Val-Asn-Ile-Thr-Val-Lys-Gln-His-Thr-Val-Thr-Thr-Thr-Thr-Lys-Gly-Glu-Asn-Phe-Thr-Glu-(Thr-Asp-Ile-Lys)-Y; Seq. I.D. No: 35X-(Met-Cys-Ile-Thr)-Gln-Tyr-Gln-Arg-Glu-Ser-Gln-Ala-Tyr-Tyr-Gln-Arg-(Gly-Ala-Ser-Val)-Y; Seq. I.D. No: 36X-(Asn-Phe-Val-His)-Asp-Cys-Val-Asn-Ile-Thr-Ile-Lys-Gln-His-Thr-Val-Thr-Thr-Thr-Thr-Lys-Gly-Glu-Asn-Phe-Thr-Glu-(Thr-Asp-Val-Lys)-Y; or Seq. I.D. No: 37X-(Met-Cys-Ile-Thr)-Gln-Tyr-Glu-Arg-Glu-Ser-Gln-Ala-Tyr-Tyr-Gln-Arg-(Gly-Ser-Ser-Met)-Y.


25. A subfragment of a synthetic polypeptide as claimed in claim 23having the sequence Seq. I.D. No: 50Val-Asn-Ile-Thr-Val-Lys-Gln-His-Thr-Val-Thr-Thr-Thr-Thr-Lys-Gly-Glu-Asn-Phe-Thr-Glu-Gly-Cys; or Seq. I.D. No: 48Cys-Ile-Thr-Gln-Tyr-Gln-Arg-Glu-Ser-Gln-Ala-Tyr- Tyr-Gln-Arg.


26. A synthetic polypeptide as claimed in claim 1 having a sequence ofgeneral Formula (IX): [L _(a) −F] _(m) −[L _(b) −G] _(n) −L _(c)  (IX)wherein F and G are each independently a polypeptide according to anyone of Formulae I to IV and VI to VIIIb, L is a linking sequence, a, band c are each independently 0 or 1, and m and n are each positivenumbers, or F and G are each independently a subfragment of suchsequence.
 27. A synthetic polypeptide which comprises an antigenicallysignificant subfragment and/or antigenically significant variant of theabove-identified polypeptide sequences as claimed in claim
 1. 28. Asynthetic polypeptide as claimed in claim 1 additionally comprising aT-cell epitope.
 29. A synthetic polypeptide as claimed in claim 1wherein X or Y comprises a retro-inverso amino acid.
 30. A syntheticpolypeptide as claimed in claim 1 linked to a carrier molecule.
 31. ADNA molecule comprising a sequence coding for at least one syntheticpolypeptide as claimed in claim
 1. 32. A vaccine comprising at least onepolypeptide as claimed in claim 1 effective to promote prophylaxisagainst encephalopathies.
 33. A kit for detecting prion proteins orantibodies against prion proteins which comprises at least one syntheticpolypeptide as claimed in claim
 1. 34. A pharmaceutical compositioncontaining as active ingredient, at least one polypeptide as claimed inclaim 1 in association with one or more pharmaceutically acceptableadjuvants, carriers and/or excipients.
 35. A method of therapy orprophylaxis of mammalian encephalopathies and/or of stimulating themammalian immune system and/or of blocking the cellular binding oraggregation of the prion proteins, which comprises administering anamount of a polypeptide as claimed in claim 1, either in isolation or incombination with other agents for the treatment of encephalopathies. 36.A method of detecting prion protein or antibodies against prion proteinor antigen binding fragments thereof, which comprises incubating asample with at least one polypeptide as claimed in claim
 1. 37. A methodof discriminating between PrP^(C) and PrP^(SC) in which a sample iscontacted with a substance selected from peptide sequences as claimed inclaim 1 preferably those relating to regions A, B and C, and significantsub-fragments thereof, antibodies raised against said sequences andsub-fragments and the presence or absence of PrP^(SC) is determined. 38.A process for the manufacture of a synthetic polypeptide having at leastone antigenic site of a prion protein, the process comprising the stepsof coupling the residues using chemical, biological or recombinanttechniques known per se and isolating the polypeptide as defined inclaim
 1. 39. An antibody or antigen binding fragment thereof whichspecifically binds to a synthetic polypeptide as claimed in claim
 1. 40.A kit for detecting prion proteins or antibodies against prion proteinswhich contains an antibody or antigen binding fragment thereof, asclaimed in claim
 39. 41. A pharmaceutical composition comprising, asactive ingredient, an antibody or antigen binding fragment as claimed inclaim 39 in association with one or more pharmaceutically acceptable,carriers and/or excipients.
 42. A method of therapy or prophylaxis ofmammalian encephalopathies which comprises administering an antibody orantigen binding fragment as claimed in claim
 39. 43. A method detectingprion proteins or antibodies against prion proteins which comprisesincubating a sample with an antibody or antigen binding fragment asclaimed in claim
 39. 44. An anti-idiotypic antibody raised against anantibody or antigen binding fragment as claimed in claim
 39. 45. Aprocess for the manufacture of an antibody which specifically binds to asynthetic polypeptide having at least one antigenic site of a prionprotein, the process comprising immunizing a non-human mammal with saidpolypeptide and isolating the antibody as defined in claim 39.